Biocompatible carbon based electrode and its preparation process

ABSTRACT

A biocompatible carbon based electrode, and its preparation process are described. The electrode is formed by first and second biocompatible chemically oxygen terminated or H-terminated carbon-based materials. The first material is configured to promote the growth or at least the direct interfacing of adult neurons on the first material without substantially promoting the growth and direct interfacing of glial cells on the first material. The second material presents a peptide coating to promote the growth and at least the direct interfacing of adult glial cells.

The present invention relates to new biocompatible carbon basedelectrode, its use and preparation process.

Neuroprostheses and brain-machine interfaces have witnessed anexponential development since the success of cochlear implants and ofdeep brain stimulation for Parkinsonian patients (Lebedev and Nicolelis,2006). Neuroprostheses can either be surface electrodes, meaning theelectrodes are simply in contact to the glial surface of the neuronaltissue such as the cortex, or penetrating electrodes reaching deeperneuronal structures in the brain. Although cortical implants have showna gain of function after a few months, this was unfortunately frequentlyfollowed by a complete loss of activity (Dobelle et al., 1974). Thisloss of functionality could be attributed to a major gliosis occurringaround the prostheses or its electrodes in the months followingimplantation (Maynard et al., 2000). Another challenge forneuroprostheses is to increase the resolution of individual electrodesto stimulate more precisely discrete neuronal areas. These objectivesmotivate the development for new biocompatible materials limiting glialreactions and improving direct interactions with neurones.

Recently, the concept of retinal prostheses was validated in clinicaltrials showing that such prostheses can enable blind patients to readshort words, identify contrasted objects or follow lanes on the ground(Humayun et al., 2009; Zrenner et al., 2011). These retinal prosthesesaiming at restoring vision in patients having lost their photoreceptorsare either placed in the subretinal space or on the epiretinal side indirect contact to either the outer or the inner limiting membrane bothproduced by glial Muller cells. In the first configuration, thesubretinal implant will stimulate retinal bipolar cells, neuronesnormally postsynaptic to photoreceptors (Zrenner et al., 2011) whereasepiretinal implants are targeting retinal ganglion cells, spikingneurones sending visual information to the brain via their axon throughthe optic nerve (Humayun et al., 2009). Although these implants havealready restored some visual functions in patients (Humayun et al.,2009; Zrenner et al., 2011), an increase in electrode resolution isrequired to further improve the restored visual performances to achieveface recognition, text reading or independent locomotion. Different 3Ddesign were already proposed to reach this objective using eitherpillars (Butterwick et al., 2009) or wells (Djilas et al., 2011) on theimplants.

Among potential new biomaterials, diamond has raised great attention forits use in neuroprostheses because Boron-doped diamond exhibitssemiconductive properties. Diamond biocompatibility was alreadydemonstrated on osteoblast cultures (Grausova L, 2009) and evenembryonic cortical neurons (Specht et al., 2004; Thalhammer et al.,2010). Embryonic neurons were found to grow selectively on proteinpatterns stamped on diamond surfaces (Specht et al., 2004) but theycould not grow directly on a polished polycrystalline diamond layer oron a nanocrystalline diamond layer unless these layers were coated withpeptides or with dispersed nanodiamond particles (Thalhammer et al.,2010). The use of diamond as electrodes has further been demonstrated byrecording neuronal activities using cell lines (Ariano et al., 2009).

One of the aims of the present invention is the use of new biocompatiblecarbon based electrodes to either promote the growth of adult neurons orincrease glial cell adherence.

Another aim is to provide such new biocompatible carbon basedelectrodes.

Still another aim is to provide a process preparation of saidelectrodes.

Still another aim is the use of such electrodes for the implementationof implants or neuroprosthesis liable to prevent the gliosis.

The present invention provides therefore:

A first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, bound to a biocompatible second chemically oxygenterminated or H-terminated carbon-based material, said first and saidsecond carbon-based material forming an electrode, said first materialbeing in particular selected from the group consisting ofnanocrystalline diamond which is doped by either boron, phosphorus ornitrogen, to become a semiconductor, or graphene, nanotubes, ornanotubes on diamond, the surface of which is substantially free of anypeptide coating, said first carbon-based material having an electricalconductivity of at least about 0.01 S·cm¹, said second biocompatiblechemically oxygen terminated or H-terminated carbon-based material,being identical or different from said first carbon-based material, saidsecond carbon-based material presenting a peptide coating, in particulara peptide from the extracellular matrix, to promote the growth and atleast the direct interfacing of adult glial cells, said secondchemically oxygen terminated or H-terminated carbon-based material beingin particular selected from the group consisting of nanocrystallinediamond which is doped in particular with boron, in particular aboron-doped hydrogen terminated diamond or oxidized diamond, graphene ornanotubes, in particular diamond grafted nanotubes, for its use topromote the growth or at least the direct interfacing of adult neuronson said material without substantially promoting the growth and directinterfacing of glial cells on said material.

An other object provided is the first biocompatible chemically oxygenterminated or H-terminated carbon-based material, bound to abiocompatible second chemically oxygen terminated or H-terminatedcarbon-based material, said first and said second carbon-based materialforming an electrode, for its use as described above, wherein said adultneurons are found in tissues selected from the group consisting of theretina, thalamus, cortex, vestibular system, cochlea, brain stem,midbrain, colliculus, subthalamic nucleus, globus pallidus interna, zonaincerta, pallidofugal fibers, periaqueductal gray, periventricular gray,internal capsule, ventral posterolateral nucleus and ventralposteromedial nucleus, subgenual cingulate gyrus, nucleus accumbens,ventral capsule/ventral striatum, inferior thalamic peduncle, lateralhabenula vagus nerve, afferent nerves, spinal cord, large dorsalcolumns, nerves controlling muscle activity such as those for locomotionor sphincter opening.

An other object provided is the first biocompatible chemically oxygenterminated or H-terminated carbon-based material, bound to abiocompatible second chemically oxygen terminated or H-terminatedcarbon-based material, said first and said second carbon-based materialforming an electrode, for its use as described above, wherein saidelectrode is shaped like a penetrating electrode, such as a penetratingneedle, in particular a tri-dimensional needle, or is positioned on apenetrating support, which can be shaped like a needle, in particular atri-dimensional needle, and in particular, a plurality of electrodesbeing combined to form an electrode array, and more particularly saidelectrode or electrode array is fixed on a support to form an implant ora prosthesis, in particular wherein the length of said penetratingneedle and/or electrode is comprised from about 10 μm to about 1 to 2 cmand the diameter is comprised from 20 μm to about 500 μm.

An other object provided is the first biocompatible chemically oxygenterminated or H-terminated carbon-based material, bound to abiocompatible second chemically oxygen terminated or H-terminatedcarbon-based material, said first and said second carbon-based materialforming an electrode, for its use as described above, wherein said firstchemically oxygen terminated or H-terminated carbon-based materialconstitutes the tip of an individual penetrating electrode or thepenetrating electrode tips of electrode arrays and said secondcarbon-based material constitutes the interfacing surface between theprosthesis or implant and the glial surface of any neuronal structure.

An other object provided is the first biocompatible chemically oxygenterminated or H-terminated carbon-based material, bound to abiocompatible second chemically oxygen terminated or H-terminatedcarbon-based material, said first and said second carbon-based materialforming an electrode, for its use as described above, wherein saidpeptide is from the extracellular matrix, in particular selected fromthe group consisting of poly-lysine, in particular poly-D-lysine,poly-ornithine, laminin or combination thereof, in particular thethickness of the peptide coating being comprised from about 0.5 μg/cm²to about 5 μg/cm².

The present invention provides a process of preparation of an electrodeor needle as described above, comprising the following steps:

a. preparation of conductive biocompatible chemically oxygen terminatedor H-terminated carbon-based material scaffold,b. optionally, oxidation of said H-terminated carbon-based materialscaffold to obtain a first carbon-based material,c. optionally doping a second part of said chemically oxygen terminatedor H-terminated carbon-based material scaffold, in particular withboron, phosphorus or nitrogen, to obtain a partially doped second partof said chemically oxygen terminated or H-terminated carbon-basedmaterial scaffold,d. coating said second part of said chemically oxygen terminated orH-terminated carbon-based material scaffold with a peptide, optionallydoped, in particular a peptide from the extracellular matrix, to obtaina second chemically oxygen terminated or H-terminated carbon-basedmaterial, said first chemically oxygen terminated or H-terminatedcarbon-based material and said second chemically oxygen terminated orH-terminated carbon-based material constituting an electrode, inparticular said peptide is selected from the group consisting ofpoly-lysine, in particular poly-D-lysine, poly-ornithine or laminin.

The present invention also provides an electrode according to the abovedescription, wherein said electrode is:

-   -   (i) a planar array with a common counter electrode with the        shape of a grid surrounding the stimulating electrodes,    -   (ii) a planar electrode array with a distant counter electrode,    -   (iii) a three-dimensional electrode, in particular a        three-dimensional electrode array with a simulation electrode        surrounded by a grid again serving as a counter electrode; or    -   (iv) a three-dimensional electrode with a distant counter        electrode with a inter-electrode distance was kept at 100 μm and        the well depth for the 3D models was 30 μm.

The present invention relates to the use of a first biocompatiblechemically oxygen terminated or H-terminated carbon-based material, thesurface of which is substantially free of any peptide coating, topromote the growth or at least the direct interfacing of adult neuronson said material without substantially promoting the growth and directinterfacing of glial cells on said material, said carbon-based materialhaving an electrical conductivity of at least about 0.01 S·cm⁻¹, inparticular 1 S·cm⁻¹, preferably 10 S·cm⁻¹, more preferably 100 S·cm⁻¹.

The expression “carbon-based material” refers to a material constitutedby only carbon atoms above 95%, the remaining percents corresponding todoping species if necessary.

“H-terminated carbon-based material” refers to carbon-based material asgrown and “oxygen terminated carbon-based material” refers to chemicallyoxidized carbon-based material.

To provide diamond with semiconducting properties, third elements can beused in its matrix such as boron, phosphorous or nitrogen, atconcentrations between 1 ppm and 5%. The term “biocompatible” refers tothe ability of a material to perform with an appropriate host responsein a specific situation without interfering or degrading the biologicalmedium (in particular a living biological medium) in which it is used.It also refers to a non immunogenic material.

The material is named “first” because it represents a first part of amaterial that can be bound to another part that will be named “second”part.

In all the specifications, the expression “a first biocompatiblechemically oxygen terminated or H-terminated carbon-based material” canalso be named “first part of a carbon-based material” or “first part ofa material” or “first part” alone and have the same meaning

The expression “substantially free of any peptide coating” means thatthe material does not present a peptide coating or if present thispeptide coating, in particular a peptide from the extracellular matrix,is in a ratio less than 1% in surface coverage.

The expression “promoting the growth and direct interfacing of adultneurons” means that the material allows either the neurons to develop onsaid material or if neurons cannot develop, at least, the materialallows the adhesion of said neurons on said material.

By adult neurons, it is referred to neurons previously collected fromanimals older than 1 months while embryonic neurons are previouslycollected on embryos and area therefore still not mature.

The term “adult” can also be replaced by “mature” or “differentiated” inthis specification.

The expression “without substantially promoting the growth and directinterfacing of glial cells on said material” means that said materialpresents a ratio of glial cells grown on it or adhered on it of lessthan 5% in surface coverage.

The material used in the invention exhibits electrical conductivity andtherefore, cannot be constituted of glass exclusively.

Said conductivity is at least 0.01 S·cm⁻¹ or is equal or higher than0.01 S·cm⁻¹, in particular said conductivity is 1 S·cm⁻¹ andadvantageously is 10 and more advantageously is 100 S·cm-1 or higher formaterial such as diamond.

In an advantageous embodiment, said conductivity is comprised from 10S·cm⁻¹ to 1000 S·cm⁻¹, preferably from 100 S·cm⁻¹ to 500 S·cm⁻¹ and morepreferably is equal to 100 S·cm⁻¹.

However, for a material such as graphene, the conductivity is muchhigher and can be as high as about 1^(E)4 S·cm⁻¹.

The inventors have unexpectedly found that a conductive carbon-basedmaterial that is not coated with a peptide, could both allows the growthor adhesion or interfacing of adult neurons on it, as well as alsoprevents the growth or adhesion or interfacing of glial cells on saidmaterial, avoiding thus the gliosis, that is the proliferation of glialcells, and the loss of functionality of said material, in particular thestimulating function of neurons by said material thanks to itsconductivity.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, wherein said material isselected from the group consisting of nanocrystalline diamond which isdoped by either boron, phosphorus or nitrogen, to become asemiconductor, or graphene, nanotubes, or nanotubes on diamond.

By “nanocrystalline diamond” is meant a layer which composition is purediamond exhibiting a grain structure, where the grain size rangesbetween 5 and 100 nm in average.

By “polycrystalline diamond” is meant a layer which composition is purediamond exhibiting a grain structure, where the grain size rangesbetween 100 nm and 50 micrometers.

By “graphene” is meant an insulated one-atom-thick planar sheet ofsp2-bonded carbon atoms that composes graphite.

By “nanotubes” is meant a hollow cylindrical molecule made of sp2-bondedcarbon atoms that composes graphite, rolled over itself to form acylinder. Nanotubes can be composed by one (single wall) or several(multiwall) carbon tubes with effect on their electronic properties.

By “nanotubes on diamond” is meant a structure where nanotubes are grownon a diamond substrate.

Materials such as diamond are not semiconductor as such and necessitateto be doped to become semiconductor. However, materials such as grapheneare semiconductor in themselves and do not necessitate to be doped.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, wherein said first chemicallyoxygen terminated or H-terminated carbon-based material is differentfrom nanodiamond powder, in particular on nanocrystalline diamond or onpolycrystalline diamond or on other materials, or from nanocrystallinediamond within which nanotubes are partially embedded.

In this embodiment, material such as powder of monodispersed nanodiamondpowder or nanocrystalline diamond within which nanotubes are partiallyembedded are therefore excluded from the scope of the invention in someaspects.

Nevertheless, it must be noted that nanotubes on diamond that aredifferent from nanocrystalline diamond within which nanotubes arepartially embedded, are not excluded form the scope of the invention.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, wherein said adult neurons arefound in tissues selected from the group consisting of the retina,thalamus, cortex, vestibular system, cochlea, brain stem, midbrain,colliculus, subthalamic nucleus, globus pallidus interna, zona incerta,pallidofugal fibers, periaqueductal gray, periventricular gray, internalcapsule, ventral posterolateral nucleus and ventral posteromedialnucleus, subgenual cingulate gyrus, nucleus accumbens, ventralcapsule/ventral striatum, inferior thalamic peduncle, lateral habenulavagus nerve, afferent nerves, spinal cord, large dorsal columns, nervescontrolling muscle activity such as those for locomotion or sphincteropening.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, wherein said first carbon-basedmaterial is bound to a biocompatible second chemically oxygen terminatedor H-terminated carbon-based material, similar to, identical to ordifferent from said first carbon-based, said first and said secondcarbon-based material forming an electrode, said second carbon-basedmaterial presenting a peptide coating, in particular a peptide from theextracellular matrix, to promote the growth and at least the directinterfacing of adult glial cells.

The second carbon-based material corresponds thus to the second partabove defined and the binding of the first part as defined above and thesecond part as defined above constitutes an electrode.

In all the specification, the expressions “a second biocompatiblechemically oxygen terminated or H-terminated carbon-based material” canalso be named “second part of a carbon-based material” or “second partof a material” or “second part” alone and have the same meaning.

Said first and second parts can be similar or identical with respect tothe material, that is they are constituted by the same material, withthe proviso that the first part is always not coated with a peptidecoating and the second part is always coated with a peptide coating, andprovided that the electrode thus constituted could still be conductive.

Said first and second parts can be different with respect to thematerial, that is they are constituted by the different materials, withthe proviso that the first part is always not coated with a peptidecoating and the second part is always coated with a peptide coating, andprovided that the electrode thus constituted could be still conductive.

The inventors have thus found that the binding of the two parts of acarbon-based material, similar, identical or different, could both,

1) promote, due to the first part, the growth or adhesion or interfacingof adult neurons on it, while preventing the growth or adhesion orinterfacing of glial cells on said first part, avoiding thus thegliosis, that is the proliferation of glial cells, and the loss offunctionality of said first part,2) promote, due to the second part, the growth of glial cells locally inthe environment of said second part but not in the first part, leadingthus to a synergy between the two parts of said carbon-based material asglial cells are essential for the survival of neurons.

In an advantageous embodiment, said first carbon-based material bound tosaid second carbon-based material is stable for at least six months.

The term “stable” means that said first carbon-based material bound tosaid second carbon-based material always constitute a functionalelectrode after six months or more.

Another advantage of the invention is to provide an electrode having alife time higher than the one of the electrodes of the prior art due tothe constitution in both parts of said electrode, avoiding thus thegliosis that is likely to occur to reconstitute the continuity in theexternal glial barrier preventing any tissue change in ionichomeostasis, and the loss of the electrode functionality that leads tothe frequent change of the electrode in the prior art.

Both first and second parts of material can be laid on a soft support(polyimide, parylene . . . ) or a rigid substrate made of silicon,glass, quartz or metal substrates (Platinum alloys or Titanium allows,Gold or Titanium, silicon carbide, . . . ).

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, having a first carbon-basedmaterial bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, similar to, identical to ordifferent from said first carbon-based, wherein said second chemicallyoxygen terminated or H-terminated carbon-based material is selected fromthe group consisting of nanocrystalline diamond which is doped inparticular with boron, in particular a boron-doped hydrogen terminateddiamond or oxidized diamond, graphene or nanotubes, in particulardiamond grafted with nanotubes.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, having a first carbon-basedmaterial bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, similar to, identical to ordifferent from said first carbon-based, wherein said first carbon-basedmaterial and said second carbon-based material are similar or identical.

Thus in this embodiment, both parts of the carbon-based material areconstituted with the same material, the first part being not coated andthe second part being coated.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, having a first carbon-basedmaterial bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, similar to, identical to ordifferent from said first carbon-based, wherein said electrode is shapedlike a penetrating electrode, such as a penetrating needle, inparticular a tri-dimensional needle, or is positioned on a penetratingsupport, which can be shaped like a needle, in particular atri-dimensional needle.

By “penetrating electrode” is meant an electrode liable to penetrate atissue such as a tissue defined above.

As an example, said electrode can penetrate the retina in order tostimulate the neurons of the retina in which photoreceptors have beendegenerated in case of pathologies such as age-related maculardegeneration (AMD) or retinitis pigmentosa.

A penetrating electrode in the prior art has the disadvantage ofdestroying the glial barrier of the tissue in which the electrodepenetrates because neuronal tissues are always surrounded by a glialcell layer acting as a physical barrier.

Another advantage of the invention is to provide a penetrating electrodethat solves this problem as the electrode is constituted of a secondpart liable to promote the growth of glial cells and thus holes createdby the penetrating needles or electrodes can be rapidly sealed.

An adequate sealing at the basis of the electrode prevents furthergliosis around the electrode tips where stimulating electrodes aredisplaced. Direct contact or close distance between neurons andelectrodes will provide the best electrical stimulation with the minimumcurrent intensities.

Said electrode can be a needle.

In an advantageous embodiment, a needle or an electrode is tridimensional. For the purpose of the present invention, the terms “tridimensional” means that the said needle or electrode is not planar orflat and is defined according to three dimensions: length, and thickness(width and height). A tri dimensional (3D) device means a device with athickness (e. g. a width) of more than 2 micrometer.

In the present invention, an electrode or a needle may have the shape ofa cylinder or of a polytope. A polytope is a finite region ofn-dimensional space bounded by hyperplanes.

In particular, a polytope is a three-dimensional figure formed by atleast six parallelograms.

In particular embodiments said polytope or cylinder is planar of flat,said needle or said electrode being tri dimensional and is square orrectangular with the greatest length comprises from 5 μm to 1 mm and oneof the thicknesses is less than 2 μm.

In a particular embodiment, said electrode is defined according to itslength and diameter.

In another advantageous embodiment, said electrode could be a planarelectrode, or a planar needle. It is well understood that a flat or aplanar electrode or a flat or a planar needle means a three-dimensionalfigure wherein the dimension defining (width) thickness is less than 2micrometer or from 2 to 0.001 micrometer.

In particular embodiments the present invention provides:

(i) a planar array with a common counter electrode with the shape of agrid surrounding the stimulating electrodes,(ii) a planar electrode array with a distant counter electrode,(iii) a three-dimensional electrode array with a simulation electrodesurrounded by a grid again serving as a counter electrode; and(iv) a three-dimensional electrode with a distant counter electrode witha inter-electrode distance was kept at 100 μm and the well depth for the3D models was 30 μm.

Electrodes according to the invention are either circular, oval, squareor rectangular with the greatest length comprises from 5 μm to 1 mm.

Electrodes are either circular, oval, square or rectangular with thegreatest length comprises from 5 μm to 1 mm.

Implants are planar whenever one of their thicknesses is below 2 μm.Implants are considered tridimensional (3D) as soon as thicknesses aregreater than 2 μm. Δ thickness difference can separate the stimulatingelectrodes and either the reference electrodes or the ground grid. Itcan also represent walls separating electrodes and the ground surfaces.The thickness difference is greater than 2 μm and usually in a rangefrom 20 μm to 1 mm although this thickness difference can reach severalcentimeters when target structures are deep inside the organ, as in thebrain. The 3D structure can take different shapes from pillars, arrows,needles, wells, cavities with a cubic, polygonal, cylindrical or conicshape with all possible combinations and intermediates.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, having a first carbon-basedmaterial bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, identical to or different from saidfirst carbon-based, said electrode being shaped like a penetratingelectrode, such as a penetrating needle, in particular a tri-dimensionalneedle, wherein a plurality of electrodes is combined to form anelectrode array.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, having a first carbon-basedmaterial bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, similar to, identical to ordifferent from said first carbon-based, said electrode being shaped likea penetrating electrode, such as a penetrating needle, in particular atri-dimensional needle, wherein a plurality of electrodes is combined toform an electrode array, wherein said electrode or electrode array isfixed on a support to form an implant or a prosthesis.

As an example and illustration, said electrode array could ultimatelyexhibit the conformation presented FIG. 12.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, having a first carbon-basedmaterial bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, similar to, identical to ordifferent from said first carbon-based, said electrode being shaped likea penetrating electrode, such as a penetrating needle, in particular atri-dimensional needle, wherein a plurality of electrodes is combined toform an electrode array, said electrode or electrode array being fixedor not on a support to form an implant or a prosthesis, wherein saidfirst chemically oxygen terminated or H-terminated carbon-based materialconstitutes the tip of an individual penetrating electrode or thepenetrating electrode tips of electrode arrays and said secondcarbon-based material constitutes the interfacing surface between theprosthesis or implant and the glial surface of any neuronal structure.

The electrode or electrode array penetrating a tissue, the not coatedfirst part must be the tip of said electrode to stimulate the neuroneinterfacing or growing on it and the second part must be the interfacingsurface between the prosthesis or implant and the glial surface of anyneuronal structure to promote the growth of said glial cells to seal theholes formed during the penetration and help the neurones of the firstpart to grow and/or survive.

The surface implant or neuroprostheses are not necessary flat, they canpresent well shapes with electrodes and/or returning grids in either thewells and/on the upper surface.

In arrays with penetrating electrodes, this peptide-coated carbonmaterial could cover the basis of the penetrating structures (e.g.penetrating needles) where direct neurone interfacing is not intended ornot possible due to gliosis for restoring the glial sheath continuity.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, having a first carbon-basedmaterial bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, similar to, identical to ordifferent from said first carbon-based, wherein said peptide is from theextracellular matrix, in particular selected from the group consistingof poly-lysine, in particular poly-D-lysine, poly-ornithine, laminin orcombination thereof.

These peptides from the extracellular matrix play a supporting role invivo that is used in vitro to promote cell attachment and thereforesurvival. The presence of these peptides is particularly useful forglial cell maintenance.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, having a first carbon-basedmaterial bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, similar to, identical to ordifferent from said first carbon-based, as defined above, wherein thethickness of the peptide coating is comprised from about 0.5 μg/cm² toabout 5 μg/cm².

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, having a first carbon-basedmaterial bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, similar to, identical to ordifferent from said first carbon-based, said electrode being shaped likea penetrating electrode, such as a penetrating needle, in particular atri-dimensional needle, as defined above, wherein the length of saidpenetrating needle and/or electrode is comprised from about 10 μm toabout 1 to 2 cm and the diameter is comprised from 20 μm to about 500μm.

In function of the tissue in which the penetrating electrode must beinstalled, the length of said penetrating electrode will be different.

As an example, the length of said electrode for cochlea is about 1 mmand for nerves controlling muscle activity such as those for locomotionor sphincter opening is about 1 cm.

In an advantageous embodiment, the present invention relates to the useof a first biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, as defined above, having a first carbon-basedmaterial bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, similar to, identical to ordifferent from said first carbon-based, said electrode being shaped likea penetrating electrode, such as a penetrating needle, in particular atri-dimensional needle, as defined above, wherein the length of saidpenetrating needle and/or electrode is comprised from about 10 μm toabout 1 to 2 cm and the diameter is comprised from 20 μm to about 500μm, as defined above, wherein the length ratio: first carbon-basedmaterial/second carbon-based material is comprised from 0.1 to 10.

In another aspect, the present invention relates to an electrodecomprising a first chemically oxygen terminated or H-terminatedcarbon-based material to promote the growth or at least the directinterfacing of adult neurons, without substantially promoting the growthand direct interfacing of glial cells on said material, which is boundto a second biocompatible chemically oxygen terminated or H-terminatedcarbon-based material, similar to, identical to or different from saidfirst carbon-based material, such as defined above, said secondcarbon-based material presenting a peptide coating, in particular apeptide from the extracellular matrix, to promote the growth and atleast the direct interfacing of adult glial cells.

In an advantageous embodiment, the present invention relates to anelectrode as defined above, wherein said second chemically oxygenterminated or H-terminated carbon-based material presents a peptidecoating, in particular a peptide from the extracellular matrix topromote the growth of adult glial cells.

In an advantageous embodiment, the present invention relates to anelectrode as defined above, wherein said electrode is non cytotoxic.

Neither the materials used, nor the doping or the coating are cytotoxicand thus all the compounds constituting said electrode are biocompatibleand not immunogenic.

In an advantageous embodiment, the present invention relates to anelectrode as defined above, which is stable for at least six months.

The term “stable” means that the electrode always functions after sixmonths or more, that is to say that gliosis has not altered saidelectrode and said electrode is always liable to stimulate adult neuronsof said tissues.

In an advantageous embodiment, the present invention relates to anelectrode as defined above, wherein said first and second carbon-basedmaterials are selected from the group consisting of nanocrystallinediamond which is doped by either boron, phosphorus or nitrogen, tobecome a semiconductor, or graphene, nanotubes, or nanotubes on diamond.

In an advantageous embodiment, the present invention relates to anelectrode as defined above, wherein said first chemically oxygenterminated or H-terminated carbon-based material and said secondchemically oxygen terminated or H-terminated carbon-based material aresimilar or identical.

In an advantageous embodiment, the present invention relates to anelectrode as defined above, wherein said electrode is shaped like apenetrating electrode, such as a penetrating needle, in particular atridimensional needle, or is positioned on a penetrating support, whichcan be shaped like a needle, in particular a tri-dimensional needle.

In an advantageous embodiment, the present invention relates to anelectrode as defined above, wherein a plurality of electrodes iscombined to form an electrode array.

In an advantageous embodiment, the present invention relates to anelectrode as defined above, wherein said electrode or electrode array isfixed on a support to form an implant or a prosthesis.

In an advantageous embodiment, the present invention relates to anelectrode wherein said electrode is shaped like a penetrating electrode,or wherein a plurality of electrodes is combined to form an electrodearray, as defined above, wherein said first carbon-based materialconstitutes the tip of an individual penetrating electrode or thepenetrating electrode tips of electrode arrays and said secondcarbon-based material constitutes the interfacing surface between theprosthesis or implant and the glial surface of any neuronal structure.

In an advantageous embodiment, the present invention relates to anelectrode, as defined above, wherein said peptide is selected from thegroup consisting of poly-lysine, in particular poly-D-lysine,poly-ornithine or laminin.

In an advantageous embodiment, the present invention relates to anelectrode, as defined above, wherein the thickness of the peptidecoating is comprised from about 0.5 μg/cm² to about 5 μg/cm².

In an advantageous embodiment, the present invention relates to anelectrode, as defined above, wherein the length of said penetratingneedle and/or electrode is comprised from about 10 μm to about 1 to 2 cmand the diameter of said electrode is comprised from 20 μm to about 500μm.

In an advantageous embodiment, the present invention relates to anelectrode, as defined above, wherein the length ratio: firstcarbon-based material/second carbon-based material is comprised from 0.1to 10.

In an advantageous embodiment, the present invention relates to anelectrode, as defined above, wherein said first chemically oxygenterminated or H-terminated carbon-based material is different fromnanodiamond powder, in particular on nanocrystalline diamond or onpolycrystalline diamond or on other materials, or from nanocrystallinediamond within which nanotubes are partially embedded.

In this embodiment, material such as powder of monodispersed nanodiamondpowder or nanocrystalline diamond within which nanotubes are partiallyembedded are therefore excluded from the scope of the invention in someaspects.

Nevertheless, it must be noted that nanotubes on diamond that aredifferent from nanocrystalline diamond within which nanotubes arepartially embedded, are not excluded form the scope of the invention.

In another aspect, the present invention relates to a process ofpreparation of an electrode as defined above, comprising the followingsteps:

-   -   a. preparation of conductive biocompatible chemically oxygen        terminated or H-terminated carbon-based material scaffold,    -   b. optionally, oxidation of said H-terminated carbon-based        material scaffold to obtain a first carbon-based material,    -   c. optionally doping a second part of said chemically oxygen        terminated or H-terminated carbon-based material scaffold, in        particular with boron, phosphorus or nitrogen, to obtain a        partially doped second part of said chemically oxygen terminated        or H-terminated carbon-based material scaffold,    -   d. coating said second part of said chemically oxygen terminated        or H-terminated carbon-based material scaffold with a peptide,        optionally doped, in particular a peptide from the extracellular        matrix, to obtain a second chemically oxygen terminated or        H-terminated carbon-based material, said first chemically oxygen        terminated or H-terminated carbon-based material and said second        chemically oxygen terminated or H-terminated carbon-based        material constituting an electrode.

The first step a. is carried out depending on the material used and ifit necessitates to be doped to become conductive such as diamond.

Thus, in the case of diamond doping is carried out at the same time, inparticular with boron, phosphorus or nitrogen.

In the case of graphene, doping is not carried out.

Diamond synthesis is commonly performed using CVD (chemical vapourdeposition) reactors where a microwave or RF or hot filament excitationenables the dissociation of species in a gas phase to initiate thegrowth of diamond on a hot substrate. The technique requires precursorgases, usually a carbon supply (eg. methane) as well as hydrogen and/orArgon. The growth occurs typically between 500 and 1000° C., thus toohigh for conventional growth on soft polymer substrates, as usuallypreferred for the fabrication of soft medical prosthesis implants. Tofabricate soft implants, the technique thus requires to deposit CVDdiamond on a rigid substrate made of silicon, glass, or metalsubstrates, on which a sacrificial layer is prepared (glass, aluminium,or other materials can successfully be used). After growth of thediamond layers, and subsequent patterning of its growth in the shape ofthe future electrodes, the wafer can be coated with subsequent polymerusing conventional low temperature processes. Then, the lift off fromthe sacrificial layer prepared prior to the diamond growth will enablethe diamond on polymer release thus the fabrication of the softimplants. This has been described in details in FR2960787.

Further, from preliminary processing of the substrate wafer, prior todiamond growth, it is possible to fabricate moulds prior to thefabrication of soft implants, thus enabling 3D geometries to beobtained. The processing of the initial wafer can take the form ofcavities, to lead to protruding diamond electrodes on the finalimplants. If the aspect ratio is high, the approach leads to 3D needlesthat are of high benefit for prosthesis electrode fabrication aspresented in this invention. Further to moulds, it is also possible tostart from 3D processed substrates exhibiting a high surface roughnessat a micrometer or lower scale (thus to enhance the electroderoughness), a technique that enables to improve the capacitance of theelectrodes due to the increase in surface ratio.

If the growth is performed using multiple steps, either overlaying ordistributed in space, and involving doped or undoped layers, it becomespossible to mix within the final diamond neuroprosthesis regions of highconductivity with regions of low conductivity.

The second step b is carried out if the material need to be oxidised toobtain a first oxygen terminated carbon-based material from a firstH-terminated carbon-based material.

The oxygen terminated surface can be prepared under various conditionssuch as annealing in air or oxygen above 400° C., or chemically in hot(>80° C.) strongly oxidizing acidic baths or also exposure to an oxygenplasma generated under RF or MW excitation.

Similarly, hydrogen termination is also prepared while exposing thecarbon surface to a short (from a few seconds to 1 hour) plasmatreatment using microwave, RF or hot filament techniques in hydrogenrich environment in order to modify the surface termination eitherH-terminated or O-terminated, according to the interests for selectivelyprepared regions that will be preferentially preferred by tissues andcell growth.

Similarly, the resulting nanocrystalline or polycrystalline diamondimplant can be used for subsequent growth of carbon nanotubes, usingconventional techniques in RF plasma reactors, or for graphene sheets tobe laid on its surface to benefit from its material properties.

In an advantageous embodiment, the present invention relates to aprocess of preparation of an electrode as defined above, wherein saidfirst part of said chemically oxygen terminated or H-terminatedcarbon-based material scaffold is doped with boron, phosphorus ornitrogen.

In an advantageous embodiment, the present invention relates to aprocess of preparation of an electrode as defined above, wherein saidpartially doped first part of chemically oxygen terminated orH-terminated carbon-based material scaffold is oxidized.

In an advantageous embodiment, the present invention relates to aprocess of preparation of an electrode as defined above, wherein saidpeptide is selected from the group consisting of poly-lysine, inparticular poly-D-lysine, poly-ornithine or laminin.

In an advantageous embodiment, the present invention relates to aprocess of preparation of an electrode as defined above, wherein saidfirst part of chemically oxygen terminated or H-terminated carbon-basedmaterial is nanocrystalline diamond, polycrystalline diamond, orgraphene.

In an advantageous embodiment, the present invention relates to aprocess of preparation of an electrode as defined above, wherein saidsecond part of chemically oxygen terminated or H-terminated carbon-basedmaterial is nanocrystalline diamond, polycrystalline diamond, orgraphene.

In an advantageous embodiment, the present invention relates to aprocess of preparation of an electrode as defined above, wherein saidfirst and/or second chemically oxygen terminated or H-terminatedcarbon-based material is different from monodispersed nanodiamond powderon nanocrystalline diamond or on polycrystalline diamond or on othermaterials or from nanocrystalline diamond within which nanotubes arepartially embedded.

In this embodiment, material such as powder of monodispersed nanodiamondpowder or nanocrystalline diamond within which nanotubes are partiallyembedded are therefore excluded from the scope of the invention in someaspects.

Nevertheless, it must be noted that nanotubes on diamond that aredifferent from nanocrystalline diamond within which nanotubes arepartially embedded, are not excluded form the scope of the invention.

In another aspect, the present invention relates to the use of anelectrode, as defined above, for the implementation of an implant or aprosthesis liable to promote the growth of adult neurons or at least thedirect interfacing of adult neurons and to stimulate said adult neurons.

In an advantageous embodiment, the present invention relates to the useof an electrode, as defined above, wherein said neurons are retinalneurons and said implant is a retinal implant.

In an advantageous embodiment, the present invention relates to the useof an electrode, as defined above, wherein said electrode furthercomprises glial cells.

DESCRIPTION OF FIGURES

FIGS. 1A to 1G present the cell labellings and cell counting of glassand diamond substrates with or without peptide coating.

FIGS. 1A to 1E present the cell labellings on glass (A-B) and diamond(C-F) substrates with (A, C, E) and without peptide coating (B, D, F).Glial cells and bipolar cells are identified in mixed retinal cellcultures, with an anti-GFAP (green: star or wire form) and an anti-Goα(red: dot form) antibody, respectively.

FIG. 1A: peptide-coated glass;

FIG. 1B: non coated glass;

FIG. 1C: peptide-coated H-(hydrogen) terminated diamond;

FIG. 1D: not coated H-terminated diamond;

FIG. 1E: peptide-coated O-(oxygen) terminated diamond;

F: not coated O-terminated diamond.

FIGS. 1G and 1H present the cell counting on the different substrates ofthe neuronal bipolar cells (G) and macroglial cells (H). Note that thedecrease in glial cells on uncoated substrates is greater than that ofbipolar cells. Scale bar, 50 μm.

x-axis: left three histograms: coated: from left to right: glass,H-diamond and 0-diamond.

Right three histograms: non coated: from left to right: glass, H-diamondand O-diamond.

y-axis: number of cells

FIGS. 2A to 2F present the survival of pure adult retinal ganglion cellneurons on O-terminated diamond and glass.

Viability of pure adult Retinal Ganglion Cell on glass (A-B) and diamondsubstrates (C-D) with (A, C) or without peptide coating (B, D).

FIG. 2A: coated glass;

FIG. 2B: not coated glass;

FIG. 2C: coated O-terminated diamond;

FIG. 2D: not coated O-terminated diamond.

FIG. 2E: Cell quantification of viable retinal ganglion cells at 6 daysin vitro normalized to their number at 1 day in vitro.

FIG. 2F: Quantification of neurite outgrowth on pure retinal ganglioncells (neurite length in μm) at 6 days in vitro.

The cell survival is significantly higher on both glass and diamondwithout any peptidic coating and the neurite outgrowth is greater onuncoated diamond and glass than on the coated substrates, verysignificantly. (means±s.e.m. from n=4 experiments, 3samples/group/experiment). Variance analysis was performed by a two-wayANOVA followed by a Bonferroni post-hoc test (***p<0.001, *p<0.05).Scale bar, 50 μm.

FIGS. 3A to 3D present the distinctive glial and neuronal preference fordiamond using peptide-patterned substrates.

Pure postnatal (p7) retinal ganglion cells (RGCs) on a peptide-patternedglass (A) or a peptide-patterned O-terminated diamond substrates (B).RGCs are revealed by their anti-NF200 immunolabelling (red fluorescence)on the peptide pattern (green: diagonal matrix for A andhorizontal-vertical matrix for B)

(C). Mixed retinal cell culture showing the glial preference for thepeptide pattern on diamond. (D) Quantification of the peptide patternsurfaces on glass and diamond with the RGC overlap on these peptidepatterns. (means±s.e.m, n=4 experiments).

x-axis: from left to right: glass, patterned glass, O-terminateddiamond, patterned O-terminated diamond

y-axis: Percentage of RGC area colocalized with the pattern:

Variance analysis was performed by a two-way ANOVA followed by a Dunnspost-hoc test (***p<0.001). The scale bar represents 50 μm.

FIGS. 4A to 4B represent a characterization of the Poly-D-lysin coatingon diamond.

FIG. 4A: Ellipsometry measurements on two H-terminated and twoO-terminated diamond samples. Variation of apparent diamond thickness(y-axis: nm) measured by ellipsometry after 45-minute incubation ofwater or poly-D-lysine (pDL).

x-axis:

left: Δ thickness (water): from left to right: O-terminated diamond;O-terminated diamond; H-terminated diamond; H-terminated diamond;

-   -   right: Δ thickness (pDL): from left to right: O-terminated        diamond; O-terminated diamond; H-terminated diamond;        H-terminated diamond;

FIG. 4B: XPS spectrum on Hydrogen-terminated diamond surface. N1s corelevel of poly-D-lysine coated H-terminated diamond. Data are expressedas means±s.e.m. from n=3 experiments.

x-axis: Binding energy (eV)

y-axis: Intensity (a. u.)

FIGS. 5A to 5H present the mixed (adult) retinal cell culture ongraphene & glass fixed at 6 days in vitro.

FIG. 5A to 5D: NBA+: from left to right with coated glass, non coatedglass, coated graphene and non coated graphene.

FIGS. 5E to 5H: NBA, 10% serum: from left to right with coated glass,non coated glass, coated graphene and non coated graphene.

Immunostaining: anti-Goalpha for bipolar cells (dot form)/anti-GFAP forglial cells (star or wire form), 3 samples for each condition

FIGS. 6A to 6H present the survival of pure adult retinal ganglion cells(8-week old) on graphene and glass, either bare or coated withpoly-D-lysine and laminin.

FIG. 6A to 6D: calcein staining, visualized by epifluorescence, of adultRGC on graphene and glass, both coated and bare;

FIGS. 6E to 6H represent, respectively, the quantification of the cellsurvival (ratio of survival between 6 div and 1 div in percentage), meancell body area (μm²), total outgrowth (μm) and total processes for bothgraphene and glass, coated and bare (from left to right).

Data are expressed as means±s.e.m. from n=4 independent experiments.

**p<0.01, *p<0.05; Two-way ANOVA followed by a Bonferroni post-hoc test.The scale bar represents 50 μm.

FIGS. 7A to 7J represent the survival of pure new-born retinal ganglioncells (p7) on graphene, glass, and sapphire either bare or coated withpoly-D-lysine and laminin.

FIGS. 7A to 7F: calcein staining, visualized by epifluorescence, of p7RGC on graphene, sapphire and glass, both coated and bare;

FIGS. 7G to 7J: quantification of the number of cells, the mean cellbody area (μm²), the total outgrowth (μm) and the total number ofprocesses for graphene, sapphire and glass, both coated and bare (fromleft to right). Results are expressed as means±s.e.m. from n=4independent experiments.

***p<0.001, **p<0.01, *p<0.05; Two-way ANOVA followed by a Bonferronipost-hoc test. The scale bar represents 50 μm.

FIGS. 8A to 8F: Pure new-born retinal ganglion cells (p7) cultured onsapphire/graphene linear patterns.

Differential interference contrast (DIC) images (FIGS. 8 a-b) andfluorescence images (FIGS. 8 d-e) representing different situationsobserved for the neurite outgrowth on the graphene lines.

In the DIC images, graphene regions (10 μm-wide stripes) appear darkerthan sapphire areas (20 or 30 μm-wide stripes). DIC image (FIG. 8 c) ofcell aggregates located at the edge between patterned and unpatternedareas. The colored scanning electron microscope image (FIG. 8 f), withgraphene regions colored in red, corresponds to the region of the 3-cellaggregate marked in (FIG. 8 c) (see also FIG. 10A to 10B).

FIGS. 9A to 9D present the characterization of CVD graphene.

FIG. 9 a: Raman spectrum of the CVD graphene used in this work. Clear Gand 2D peaks are observed confirming the presence of graphene. The ratioof the 2D to the G peak and the Lorentzian shape of the peaks indicate ahigh surface coverage by single layer graphene. The small D peaksuggests a rather low defect density.

FIG. 9 b: Atomic force micrograph. The typical wrinkles are a clearindication for graphene. Punctual residual dirt from the transferprocess can still be seen.

FIG. 9 c: Scanning electron micrograph of a patterned sample.Well-defined, alternating lines of graphene (10 μm wide, bright) andbare sapphire (20 μm wide, dark) can still be seen after cell culture.

FIG. 9 d: Electrical characterization of a graphene solution-gatedtransistor. The drain-source current is plotted against the gate sourcevoltage and shows a “dip” behavior, which is typical for ambivalentcharge transport in graphene. From these measurements, mobilities higherthan 8000 cm²V⁻¹ s⁻¹ can be calculated.

FIGS. 10 a to 10 b present the pure new-born retinal ganglion cells (p7)cultured on sapphire/graphene patterns.

FIG. 10 a: Scanning electron microscope image of cell aggregates in thevicinity of the limit between patterned and unpatterned areas,corresponding to the DIC image in FIG. 8 c. The top square correspondsto the SEM image in FIG. 3 e.

FIG. 10 b: SEM image of the cell aggregate marked by the bottom squarein (10 a).

FIG. 11. Influence of the poly-D-lysine/laminin coating on theelectronic properties of graphene has been assessed. Graphenesolution-gated field effect transistors (SGFETs), fabricated asdiscussed elsewhere (Dankerl, et al., 2010), were characterized beforeand after the coating. The transistor characterization was performed inan aqueous electrolyte containing phosphate buffered saline (5 mM) at apH of 7. For all investigated transistors, an average shift of the Diracpoint of −60 mV was observed, which is in agreement with the expectedshift induced by the positively charged poly-D-lysine coating. Such ashift approximately corresponds to a charge density of 1012 charges percm². Both before and after coating the transistors reachtransconductances of 3-4 mS·V⁻¹ (normalized by the drain-sourcevoltage). This suggests that the carrier mobility remains unchangedafter coating, thus confirming that the poly-D-lysine/laminin coatingdoes not influence the electronic properties of graphene.

FIG. 12 presents an example of an implant constituted with severalelectrodes of the invention carbon-based material such as diamond, orgraphene.

Up vertical arrow corresponds to the first carbon-based material (noncoated) and down vertical arrow corresponds to the second carbon-basedmaterial (coated).

The length of each first and second carbon-based material described hereis given for example only and the invention is not limited to thislength.

First and second carbon-based material constitutes an electrode.

Such electrodes can be put together to constitute an implant or aprosthesis liable to stimulate neurons.

Thus the space between two neighbor electrodes must also be coated.

FIG. 13 represents an image of Abraham Lincoln before (A) and after (B)down sampling to 25×25 pixels and reducing color palette to 4 grayscalelevels.

FIG. 14 represents the current density plots 20 μm above the electrodearray for the four simulated models, visualized by a red line on eachrespective slide view.

A: planar with surrounding counter-electrode;

B: planar with distant counter-electrode located in the lower rightcorner;

C: three-dimensional with the counter-electrode surrounding the wells;

D: three-dimensional with a distant counter-electrode located in thesame position as in B.

FIG. 15 represents a fabrication of flexible diamond implants. A:schematic of the microfabrication steps; B: Picture of mask (Yellow: KOHstructures, Red shape of implant); C: SEM picture of Silicon Mould; D:Final dummy implant for in vivo evaluation.

FIG. 16 represents eye fundus of P23H rats implanted with polyimide (A),diamond (B) and metallic (C) devices.

FIG. 17 represents Scanning Electron Micrographs of polyimide (A, D),diamond (B, E) and metal (C, F) implants.

FIG. 18 represents confocal imaging of stained retinae in contact withimplant+quantification of bipolar cell within the 3D electrodes for eachmaterial.

FIGS. 19 (A) and (B) (from Djilas et al., 2011) represent 3D electrodegeometries and their corresponding half cross sections. The dashed linesin the 3D representations mark geometry half cross sections. Eachelectrode half cross section is defined with five parameters (p1-p5)which represent the lengths of the segments.

Cavity wall inclination is 54.7° for both geometries. Active electrodesegments (thick lines in the half cross sections) are separated byinsulated segments (thin lines in the half cross sections).

EXAMPLES Example 1 Diamond Material Example 1-1 Diamond Growth andSamples Preparation

Nano-crystalline diamond films were produced on Corning incorporatedGlass 1737-F (6×6×1 mm³). On these glass plates, detonation diamondnano-particules (6-nm) dispersed in water (0.1% wg) were spread by spincoating. Then a 300 nm-thick nano-crystalline diamond film was grown atlow temperature in a SEKI TECHNOTRON CORP AX6500 Microwave PlasmaChemical Vapor Deposition (MPCVD) system. The growth parameters areprovided in Table 1. Then, these Hydrogen-terminated diamond surfaceswere oxidized by immersion during 30 min in a boiling solutioncontaining 98% concentrated sulphuric acid and an excess of potassiumnitrate (H₂SO₄/KNO₃). Diamond on glass substrates were reused byexposing them to a hydrogen plasma treatment. This treatment wasachieved in the same reactor that was used for diamond growth but in thecondition described in Table 2. For the current study, after growth thesamples were cut in 6×6 mm² chips in order to provide biologists withstatistically representative series of samples (typically 100) for theperforming of cell culture studies. Hydrogen-terminated diamonds werestored in a sterile petri dish just out of the reactor and were rinsedwith sterile culture medium right before seeding the cells on them.

TABLE 1 Diamond growth conditions on glass substrates MW power PressureH₂ flow CH₄ flow Temperature Duration (kW) (mbar) (sccm) (sccm) (° C.)(h) 2 30 196 4 650 4

TABLE 2 Diamond hydrogenation conditions MW power Pressure H₂ flowTemperature Duration (kW) (mbar) (sccm) (° C.) (h) 1.7 35 200 650 0.5

Example 1-2 Micro-Contact Printing of Peptide Patterns

Microstamps were produced by photolithography and molding (Mrksich etal., 1997; Chang J C, 2001). An electron beam writer transposed thegradient structures into a chrome mask. Applying UV-photolithography,master stamps were produced using 5-12.5 μm thick positive photoresist(AZ 5206) layers on 0.6 mm thick silicon wafers. Pattern transfer wasachieved by deep reactive ion etching (2.5 μm deep). Finally, the moldwas passivated by covalently linking a (trichloro (1H, 1H, 2H,2H-perfluorooctyl)silane layer to its surface in a vapor deposition(Mrksich et al., 1997) process to support an easy release of theplastomer and reuse of the mold. Poly(dimethylsiloxane) (PDMS)microstamps were fabricated curing Sylgard 184 (Dow Corning, Midland,USA) onto these masters followed by curing at 55° C. for 48 h. Aftermaster stamp release, final curing was performed for 1 h at 110° C.Inking took place by immersing the stamp for 20 min in 10 μg/mlFITC-(fluorescein isothiocyanate) conjugated poly-L-lysine (FITC-PLL)(Sigma, Germany) mixed with 2 μg/ml laminin (from mouse sarcoma, SigmaL2020) both diluted in Hank's Balance Salt Solution (HBSS). The stampwas dried in nitrogen stream and pressed for 2 minutes onto the diamondsubstrate, pre-sterilized with 70% ethanol pA.

Example 1-3 Analysis of the NCD Thin Films: Ellipsometry and XPSMeasurements

Ellipsometry and XPS measurements were used to characterize oxidized andhydrogenated diamond substrates with or without a peptide coating. Ashydrogen termination is altered by natural diamond oxidation in air,surface hydrogenation was performed just prior to the ellipsometrycharacterization in a semi-industrial Microwave Plasma Chemical VapourDeposition (MPCVD) reactor SEKI AX6500. The experimental conditionswere: microwave power 2200 W, pressure 25 mbar, hydrogen flow 200 SCCMand temperature 650° C.

A UV-Vis Spectroscopic Ellipsometer (Horiba Jobin-Yvon, UVISEL), at afixed angle of 70°, was used to characterize the roughness and thethickness of diamond films. For both oxidized and hydrogenated surfaces,the diamond sample was fixed onto the ellipsometer in order to alwayscharacterize the same area. Measures were obtained on the “bare” surface3 times, and then a 20 μl drop of either deionized water or 0.1 mg/ml ofpoly-D-Lysine in water was deposited onto the sample. After 45 min atroom temperature, the sample was thoroughly rinsed with deionized waterand dried with argon. Finally, measures were taken 3 more times on thesame area but on the “coated” sample. The use of a three-layermicro-structural model is necessary to obtain a good fit of theexperimental data (surface roughness/diamond/silicon) (Lions et al.,2009). Towards the modelling of the optical constants, they were fittedusing a Tauc-Laurentz dispersion law (Gupta et al., 2008). For both“bare” and “coated” samples, the same model was used: diamond thicknessand apparent diamond thickness were extracted, respectively. A plot ofthe difference in apparent diamond thickness once the surface is coatedwith respect to before the coating was then realised.

Surface analysis was also performed by X-ray photoelectron spectroscopy(XPS) onto those samples. The spectrometer anode supplied was providedwith a hemispherical analyser and an Al—K monochromator. Bindingenergies were referenced to the Au4f_(7/2) peak located at 83.6 eV. Acurve fitting procedure was carried out on smoothed spectra to extractthe components in the N1s spectrum using Voigt functions with aLorentzian half-width of 0.2 eV and 0.6 eV, respectively.

Example 1-4 Animals

Long-Evans rats, used for cell cultures, were purchased from Janvier (LeGenest Saint-Isle, France). All Experiments have been carried out inaccordance with the European Community Council Directives (86/609/EEC)and with the ARVO (Association for Research in Vision and Ophthalmology)statement for the Use of animals in ophthalmic and visual Research.

Example 1-5 Primary Retinal Cell Cultures Mixed Retinal Cells

Primary mixed cultures of retinal cells were isolated from retinae ofadult Long Evans rats (8 week-old). Animals were anesthetized andsacrificed; their eyes enucleated and placed in a solution ofCO₂-independent medium (Life Technologies, Carlsbad, Calif., USA).Retinae were cut in pieces of about 2 mm² and rinsed twice in Ringermedium. Retinal pieces were then incubated for 25 min at 37° C. in asolution of 2 UI/ml of papain (Worthington, Lakewood, N.J., USA),previously activated for 30 min at 37° C. in Ringer medium. They werethen rinsed with Neurobasal-A medium (Life Technologies, Carlsbad,Calif., USA) containing 10% of Foetal Bovine Serum (FBS; Sigma) and 2.5%of DNAse (DNAse, Sigma). Retinae were dissociated in this latter medium,in four steps, using 1 ml cones and adding 1 ml of the solution ofNeurobasal-A medium and 10% FBS at each step. Cell suspension wascentrifuged at 115 g during 6 min at room temperature. Supernatant wereremoved and the pellet was resuspended in Neurobasal-A medium (LifeTechnologies), which contained 2 mM L-glutamine (Life Technologies) and2% of B27 (Life Technologies). Finally, cells were counted by additionof 10% of trypan blue (Life Technologies) using a malassezhemocytometer. They were seeded in the same medium (NBA+) at an initialdensity of 2×10⁴ cells/cm² on 8 mm-diameter glass coverslips or on 6×6mm² diamond samples, either coated by successively poly-D-lysine (2μg/cm² for 45 min; Sigma-Aldrich) and laminin (1 μg/cm² overnight;Sigma-Aldrich) or uncoated.

NBA=Neurobasal medium.NBA+=NBA with 2% B27 and 1% de Glutamine.The composition of B27 is described below:

Biotine Selenium

L-carnitine T3 (triodo-1-thyronine)Corticosterone DL-α-tocopherol (Vitamin E)Ethanolamine DL-α-tocopherol acetateD (+)-galactose ProteinsGlutathione (reduced) Albumin bovineLinoleic acid InsulineLinolenic acid CatalaseProgesterone Superoxide dimutase

Putrescine Transferrin

Retinyl acetate

Pure Retinal Ganglion Cells (RGCs)

RGCs were isolated from retinae of adult Long-Evans rats (8-week old)with an immunopanning technique, according to protocols previouslydescribed in young rats (Barnes et al., 1988) and adult animals (Fuchset al., 2005a). As well as for the mixed cultures, cells were seeded atan initial density of 2×10⁴ cells/cm² on glass coverslips and diamondsamples, with or without peptide coating on the whole surface or as apattern.

Example 1-6 Viability Test

Cell viability was assessed with the “lived-dead” test (LifeTechnologies), which consists in labelling viable cells with calceinAMdetected as green fluorescence, whereas dead cells were labeled withethidium Iodide producing a red fluorescence. Briefly, coverslips wereincubated in a mixture of calceinAM and ethidium homodimer-1 (diluted ina PBS medium) for one hour in the incubator (humidified chamber, 37° C.,5% CO₂). Only alive RGCs were counted from seven fields selectedidentically on each coverslip using a microscope (Leica DM 5000B, Solms,Germany) equipped for epifluorescence. Viable RGCs were counted at 1 and6 days in vitro (DIV). When evaluating the effect of the differentmaterials, the results were presented as a percentage of cell survivalat 6 DIV with respect to 1 DIV.

Example 1-7 Immunocytochemistry and Fluorescent Imaging

Cell cultures were fixed with 4% paraformaldehyde (Sigma) in PBS (Sigma)for 15 min at room temperature (20° C.). Cell cultures were rinsed,permeabilized for 10 min in PBS containing 0.1% Triton X100 (Sigma) andpretreated with PBS containing 3% BSA (Sigma), 10% normal goat serum(Sigma-Aldrich) and 0.1% Triton X100 for 1 h to reduce non-specificlabelling. Primary antibodies were applied on the cell cultures for 2 hat room temperature (anti-G0α diluted at 1/1000 for bipolar cellsstaining and anti-GFAP diluted at 1/500 for glial cells staining) Afterseveral washes, cell cultures were incubated with a goat anti-rabbit IgGantibody conjugated to Alexa™ 488 (Molecular Probes, Eugene, Oreg., USA)and Alexa™ 594 (Molecular Probes, Eugene, Oreg., USA) diluted at 1/500for 1 h at room temperature. Nuclear labelling was achieved byincubating the cell cultures in a DAPI solution with the secondaryantibody. Cell cultures were rinsed four times with PBS-Triton 0.1%,once with PBS only and at last once with distilled water beforemounting. The fluorescent labelling was observed using a Leica DM 5000microscope with epifluorescence illumination (Alexa™ 488: excitationfilter 480±40 nm; Alexa™ 594: excitation filter 560±40 nm).

Example 1-8 Quantification: Cell Viability, Neurite Sprouting andColocalization

Density of pure cultured RGCs was evaluated by countingcalceinAM-positive viable cells at 6 days in vitro (DIV) from sevenfields selected identically on each coverslip under an epifluorescencemicroscope (Leica DM 5000B). Full glass coverslips and diamond sampleswere digitalized (˜300 fields for a 10× magnification) to automaticallyquantify (i) the neurite sprouting for the pure RGC cultures and (ii)the colocalization of cells and neurites on the protein patterns usingMetaMorph software (Ropper scientific). The <<Neurite Outgrowth>>application takes into account the diameter of the cell bodies, thewidth of the neurites and their intensities above background.

For the cell colocalization with the patterns, images of the differentmicroscope fields were thresholded and the colocalized area of bothimages (cells and pattern) was expressed as percentages.

For the mixed cultures, glial cell area was measured with Metamorphsoftware using an automatic threshold on the image, quantifying only thearea stained with GFAP.

Example 1-9 Statistical Analysis

All data are expressed as means±s.e.m. One-way ANOVA was used forvariance analysis, followed in case of significance by either aBonferroni post-hoc test (Gaussian distribution) or a Dunns post-hoctest (no Gaussian distribution) to compare the means of each group.Differences were considered significant at *p<0.05, **p<0.01 and***p<0.001.

Conclusion:

The present study further confirms that neuronal tissue cells can begrown on diamond. Here it has been demonstrated that pure adult retinalneurones can survive on uncoated diamond whereas glial cells preferpeptide coated diamond. Pure neurones even generate longer neurites onbare diamond than on peptide coated diamond. When both grounds arepresent, these neurites do not select the peptide-coated diamond muchmore significantly than the bare diamond by contrast to peptide-coatedglass versus bare glass. These results therefore highlight some hints onthe diamond use in neuroprostheses.

Diamond Biocompatibility on Neuronal Tissue Cells

Two studies had investigated this point with cultures of embryoniccortical neuronal cells (Specht et al., 2004; Thalhammer et al., 2010).In these studies, diamond layers (polished polycrystalline ornanocrystalline) appeared to provide a substrate equivalent to glass forneuronal growth. Indeed, they all allowed neuronal growth only whenpeptide coated or covered with dispersed nanodiamond particle. In thesestudies, neurons were mixed with glial cells and both cell types werenot surviving directly on the uncoated polished polycrystalline ornanocrystalline diamond layers. However, as neuronal survival was oftenreported to rely on glial cell presence or addition of glial-conditionedmedium (Fuchs et al., 2005b), it remained unclear whether the poorneuronal survival on uncoated diamond was specific or indirectly relatedto the absence of glial cell growth.

Similarly no major difference between glass and diamond for the survivalof mixed retinal cells was found in the invention. By contrast to thesestudies, both glial cells and bipolar neuronal cells on uncoatednanocrystalline diamond layers as on uncoated glass were observed.

When counting glial cells and bipolar cell neurones, the decrease inglial cells was greater than that of neurons on the uncoated diamondlayers. These results suggested that glial cells are more sensitive toculture substrates than neurons. The square shapes of glial cells on thepatterned substrates with processes elongating along the linear peptidecoating further confirmed their strong preference for the peptidecoating. Therefore, when glial cell adhesion to the material isrequired, peptide coating of diamond layers should be stronglyrecommended. For such a peptide coating on the diamond surface,H-terminated diamond have to be preferred because the peptide coating isthicker on these substrates with respect to O-terminated diamond asindicated by surface state measurements.

Dispersed nanodiamond particles appear as an alternative to peptidecoating because embryonic neuronal cultures survived very well on theseparticles (Thalhammer et al., 2010). In fact, it would be difficult toconsider solely these particles for implant coating because they are notbound to the substrates and could therefore be engulfed and moved toother tissues by phagocytes. However, the growth of embryonic neuronalcultures on these dispersed nanodiamond particles (Thalhammer et al.,2010) suggests that chemical or physical modifications of diamondsurfaces occurring during the particle preparation can change cellularinteractions with glial cells. Further studies will therefore have toinvestigate how chemical and physical modifications of diamond canmodify glial cell adherence and growth.

By contrast to previous studies using embryonic cell cultures (Specht etal., 2004; Thalhammer et al., 2010), adult neurons surviving directly onthe diamond substrates were often observed indicating that they do notrequire peptide coating at the interface with diamond or glass. However,in these mixed cultures, neurons were less dense on diamond and glassthan on the peptide coated substrates. This decrease in the number ofsurviving neurons could in fact result from the decrease in glial cellnumber because glial cells are known to release important factors forneuronal survival (Fuchs et al., 2005b). To eliminate this glialvariable, neuronal survival was assessed using pure retinal neuronsfollowing the purification of adult retinal ganglion cells. Thispreparation showed that the peptide coating did not influence neuronalcell survival and even suppressed neurite outgrowth on both glass anddiamond. These observations suggest that a direct neuron/diamondinterface is compatible with neuronal cell survival and neuritedevelopment. Such a direct interface could be very important to increasestimulation yields in neuroprostheses. Although neuronal processes werelonger on uncoated glass, they preferred to grow on the peptide-coatedglass when both grounds are available. Using embryonic neuronal cellcultures, neuronal cell processes were similarly reported to growpreferentially on the peptide-coated diamond substrates (Specht et al.,2004). In our hands, this strict preference for the peptide-coateddiamond substrates was not recovered when using pure adult retinalganglion cells. This result confirmed further that adult neurons canwell survive and grow neurites directly on diamond.

Diamond in Neuroprostheses

For neuronal stimulation, the tissue/implant interface has to be astight as possible to limit ionic diffusion at the surface of theimplant, which would limit the resolution of individual electrodes andthe current intensity to be injected for reaching the activationthreshold of the neurons. In fact, neuronal tissues are alwayssurrounded by a glial cell layer acting as a physical barrier.Therefore, neuroprostheses can either be deposited on this glial surfaceor exhibit penetrating electrode invading into the neuronal tissue. Forsurface implants, it will be important therefore to increase the glialcell adherence to the implant and electrodes. Therefore when consideringthe use of diamond electrodes, these results suggest that peptide coateddiamond has to be selected. This applies particularly for retinalimplants, which will come in direct contact to the inner limitingmembrane (epi-retinal implants) or the outer limiting membrane(subretinal implant) both formed by glial Muller cells. The outerlimiting membrane is indeed covering the retina on the photoreceptorside after the loss of photoreceptor inner and outer segments.

In case of penetrating implants, a massive glial response was found todevelop around electrodes within a few months (Maynard et al., 2000).This massive gliosis may explain the loss of electrical stimulationoccurring in a few months after implantation as in cortical prostheses.This massive gliosis is likely to occur to reconstitute the continuityin the external glial barrier preventing any tissue change in ionichomeostasis. This suggests that holes created by the penetrating needlesor electrodes should be rapidly sealed. The sealing of these holes couldbe achieved by applying a molecule promoting glial cell adhesion at thebasis of penetrating needles or on the implant socket. This materialcould be for instance any peptide-coated material like peptide coateddiamond. An adequate sealing at the basis of the electrode may preventfurther gliosis around the needle tips where stimulating electrodes aredisplaced. In fact, direct contact or close distance between neurons andelectrodes is expected to provide the best electrical stimulation withthe minimum current intensities. Therefore, our results suggest thatbare diamond without any peptide coating should be used at the needletips where stimulating electrodes are located to limit glial celladherence and growth but favor instead direct contacts to neuronalprocesses.

Conclusion

This study confirms the biocompatibility of diamond for neurones andglial cells. It therefore strengthens the need to consider diamond as anattractive electrode material for applications with stimulation of theneural tissues is sought.

Example 2 Graphene Material Example 2-1 Graphene Preparation andProcessing

Graphene Growth and Transfer.

Single layer graphene was prepared by chemical vapor deposition (CVD).After an annealing of the copper foil at 1000° C. under hydrogen flow(35 sccm) for 15 min, the gas composition was changed to ahydrogen/methane mixture (10 sccm/35 sccm) and the graphene was grown oncopper foil at 1000° C. for 30 min. As reported previously [Li, et al.,2009], a high surface coverage of single layer graphene is obtainedunder these conditions [Hess, et al., 2011]. For the wet transfer ofgraphene to sapphire, the copper/graphene films were covered with apoly(methyl 2-methylpropenoate) (PMMA) layer for transfer. This stackwas placed on a FeCl₃ solution (0.5M) to etch the copper. Afterwards,the graphene/PMMA film was transferred to sapphire substrates and thePMMA was stripped with solvents. Finally, samples were annealed in UHVin order to reduce surface contamination.

Linear Patterns of Graphene on Sapphire Substrates.

10 μm-wide graphene lines were patterned on the sapphire substrates byusing UV-photolithography and a subsequent oxygen plasma treatment.

Example 2-2 Primary Retinal Cell Cultures: Purification of RetinalGanglion Cells (RGCs)

Cell cultures were prepared from Long-Evans rats, which were purchasedfrom Janvier (Le Genest Saint-Isle, France). All Experiments have beencarried out in accordance with the European Community Council Directives(86/609/EEC) and with the ARVO (Association for Research in Vision andOphthalmology) statement for the Use of animals in ophthalmic and visualResearch.

RGCs were isolated from retinae of adult and new-born Long-Evans rat(8-week old and postnatal day 7), with an immunopanning technique,according to protocols previously described in young rats [Barres, etal., 1988] and adult animals [Fuchs, et al., 2005b]. Cells were seededat an initial density of 2×10⁴ cells.cm⁻² on glass coverslips, grapheneand sapphire samples, in Neurobasal-A medium (Life Technologies), whichcontained L-glutamine (2 mM, Life Technologies) and B27 (2%, LifeTechnologies) for adult cells and ND-G medium [Barres, et al., 1988] fornew-born, either coated by successively poly-D-lysine (2 μg·cm⁻² for 45min; Sigma-Aldrich) and laminin (1 μg·cm⁻² overnight; Sigma-Aldrich) oruncoated. The poly-D-lysine/laminin coating does not influence theelectronic properties of graphene, as discussed in the Supportinginformation.

Example 2-3 Viability Tests

Cell viability was assessed with the “lived-dead” test (LifeTechnologies), which consists of labelling viable cells with calceinAMdetected as green fluorescence, whereas dead cells were labeled withethidium Iodide producing a red fluorescence. In each experiment, boththe coated and the uncoated conditions included two glass plates, threegraphene and three sapphire substrates. After 6 days in culture, theywere incubated in a mixture of calceinAM and ethidium homodimer-1(diluted in a PBS medium) for one hour in the incubator (humidifiedchamber, 37° C., 5% CO₂). Digital images of calceinAM-positive viablecells (630 μm×750 μm) were obtained at 15 fixed independent locationsunder an epifluorescence microscope using a 10× objective (Leica DM5000B, Solms, Germany). The numbers of viable RGCs were normalized ineach experiment to those surviving at 1 day in vitro (DIV) onpeptide-coated glass. All quantifications (cell numbers, cell sizes,neurite lengths, process number) were all obtained automatically fromthe digital images using the specific applications of Metamorph (Ropperscientific). Each experiment was then repeated at four times with allthe substrates with or without coating.

Example 2-4 Mixed (Adult) Retinal Culture on Graphene and Glass Fixed at6 Days In Vitro

The method used is as in Example 1.5

Results are presented in FIGS. 5A to 5H.

Example 2-5 Statistical Analysis

All data are expressed as means±s.e.m. Two-way ANOVA test was used forvariance analysis, followed in case of significance by either aBonferroni post-hoc test to compare the means of each group. Differenceswere considered significant at *p<0.05, **p<0.01 and ***p<0.001.

Conclusion:

FIG. 6 illustrates the viability of adult retinal neurons after 6 daysin vitro, not only on peptide-coated graphene but also directly on baregraphene. All viable neurons exhibit neurite outgrowth on any substrate,peptide-coated or uncoated (FIG. 6 a-d). The survival rate wasquantified in repeated experiments and no statistically significantdifferences were observed between peptide-coated substrates, eitherglass or graphene. Surprisingly, the peptide coating did not increasethe survival rate on glass in contradiction to common views for cellcultures [Clark, et al., 1997; Fricke, et al., 2011]. On bare graphene,on the other hand, viable cells were slightly less numerous than onglass or peptide-coated substrates; such differences, even if small, arestatistically significant (see FIG. 6 e). However, despite this slightdecrease in cell number on bare graphene, the presence of neurons withoutgrown neurites on this bare substrate strongly confirms the highcytocompatibility of graphene. To further investigate any differencebetween glass and graphene substrates, several parameters including cellsize and neurite outgrowth on the different substrates were quantified.When peptide-coated, glass and graphene offered no difference. However,in the absence of coating the cell size was smaller on graphene than onglass suggesting thereby that cells had more difficulty to adhere and tospread over bare graphene (FIG. 6 f). This difference in cell adherencecould also explain why neurite lengths were significantly reduced onbare graphene as compared to all other substrates (FIG. 6 g). Inaddition, it could provide an interpretation for the apparent reductionin the process number even if the difference was not statisticallysignificant (FIG. 6 h). These observations indicate that adult neuronscan nicely survive and grow neurites directly in contact with bare (i.e.non peptide-coated) graphene.

To further assess neurite sprouting on graphene as well as on graphenepatterns, postnatal retinal ganglion cells (postnatal day 7) maintainedin a Neurobasal-based medium (containing growth factors, hormones andvitamins were used [Barres, et al., 1988]. The developmental stage ofpostnatal cells makes them particularly prone to such outgrowth [Adler,et al., 1990]. To investigate the effect of graphene patterns on cellgrowth, we also evaluated sapphire since it is used as a substrate todeposit graphene. The numerous cell bodies and the very high densitiesof cell processes observed on all substrates after 3 days (FIG. 7 a-f)are consistent with the above-discussed graphene cytocompatibility foradult neurons.

However, the peptide coating of the substrates was found to result in a2 fold increase of the apparent cell number (FIG. 7 g) and a 10 foldincrease of the apparent neurite length (FIG. 7 i). In fact, the cellmorphology was different on bare substrates with an apparent increase incell body size (FIG. 7 h), which is indicative of cell aggregation.Thus, the apparent reduction in cell number for uncoated substrates(FIG. 7 g) is partially influenced by this cell aggregation in cellclumps. Furthermore, cell processes on uncoated graphene and sapphirewere thicker suggesting the production of neurite bundles [Watkins, etal., 2008] which thus leads to the major reduction in the apparentneurite length (FIG. 7 i). This study indicates that young neurons andtheir neurites have a tendency to aggregate on bare substrates. Thepeptide coating may therefore improve cell adherence of cell bodies,neurites and their growth cones, such that in its absence all thesestructures tend to regroup.

To investigate further the potential competitive advantage of baregraphene as a substrate, postnatal retinal ganglion cells were grown onalternating linear patterns of sapphire (20 or 30 μm-wide) and grapheneon sapphire (10 μm-wide)(FIG. 8). No peptide coating was applied priorto the cell culture in order to avoid its potential masking effect ofthe substrate. Cell morphologies were precisely revealed by thefluorescent live/dead assay while differential interference contrastmicroscopy was used to localize the graphene patterns. As describedabove, the cell bodies aggregate to form cell clumps. Similarly, largeneurite bundles were seen to emerge from these cell clumps. FIG. 8illustrates different situations of neurite outgrowth on the graphenepatterns. Interestingly, the neurite bundles often follow the edge ofthe graphene patterns crossing over the lines from time to time. In someoccasions, the cell neurites appear to wander independently of thepattern (FIG. 8 a). In other cases, however, the neurite sproutingfollows very strictly the lines and could cross them (FIG. 8 b). FIG. 8e nicely shows an aggregate of 3 cell bodies adhering both on grapheneand sapphire while 2 neurites developing from this aggregate grow alongneighboring graphene lines, which is confirmed by the corresponding SEMimage shown in FIG. 8 e (see also FIG. 10). The often observed guidanceof neurites at the graphene edge (FIG. 8 b) might be due to its chemicalcomposition rather than the physical step, which height is of the orderof 1 nm. Due to the technology employed to prepare the graphene/sapphirepattern, the graphene edge is very likely to be decorated with oxygengroups, whereas far away from the edge, no such groups are expected.Such hydrophilic surface has been shown to improve neuronal viability,possibly through improved cell adhesion [Jung, et al., 2001, Liu, etal., 2006, Khan, and Newaz, 2010].

Previous studies have investigated the survival of mixed glial/neuronalcells on polylysine-coated graphene [Li, et al, 2011] or proliferatingneuronal cells (stem cells, PC12 cell line) either on laminin-coatedgraphene or incubated with graphitic nanomaterials (graphene layers)[Park, et al., 2011, Zhang, et al., 2010, Yen, et al., 2011]. Theoriginality of this work lies in the use of purified adult and/ordifferentiated retinal neurons. In addition, the survival of theseneurons was assessed on graphene directly without any peptide coating ofthe substrate. The importance of the peptide-coating on similar oridentical carbon-based materials was even demonstrated using patterns ofpeptide coating, which allowed embryonic cortical neurons to growpreferentially on the laminin coating rather than directly on thediamond [Specht, et al., 2004]. For in vitro cytocompatibility studieson graphene, the coating is likely to introduce some bias in short-termcultures. In a long-term culture or an in vivo implant, however, thecoating will end up dissolving/degrading after a couple of weeks andthis might reveal a toxicity hidden by the presence of coated proteinsmasking the graphene surface.

This is not the case in this study, because even with short-termcultures graphene is investigated both bare and coated with proteins. Asecond important novelty aspect of this work is the use of cultures ofadult neurons on graphene, which is in contrast to previous studiesreporting on cultures obtained from embryonic or new-born tissues.Aiming at testing materials for neuroprosthetic devices,cytocompatibility studies using adult neurons provide a more realisticmodel system since these prostheses are to be implanted in adultpatients.

These results demonstrates that adult neurons can survive and growneurites when cultured on bare (i.e. non-coated with peptides) graphene,confirming its great potential as a cytocompatible material forinterfacing neurons with electronic devices. In a recent development,graphene transistor arrays have been successfully used to record actionpotentials from cardiomyocytes [Hess, et al., 2011, Cohen-Karni, et al.,2010].

Example 3 Coating Protocol

A solution of poly-D-lysine at 2 μg/cm² is prepared in Phosphate BufferSaline (PBS). A drop of this poly-D-lysine solution, typically between100 μL and 1 mL depending on the size of the implant, is deposited atthe basis of the electrodes, so that the electrode tips are notimmersed, and left for 45 minutes to incubate. The basis of theelectrodes is then rinsed in PBS and a solution of laminine at 1 μg/cm²is applied also at the basis of the electrodes. The incubations stepsare done in a humidified chamber (37° C., 5% CO2).

Examples 4 Electrodes

Electrodes were prepared as previously described in Djilas et al., 2011and coated according to the above described process to provide coateddevices.

Briefly, 3D electrode geometries and their corresponding half crosssections are as illustrated in FIGS. 19 (A) and (B). The dashed lines inthe 3D representations mark geometry half cross sections. Each electrodehalf cross section is defined with five parameters (p1-p5) whichrepresent the lengths of the segments.

Cavity wall inclination is 54.7° for both geometries. Active electrodesegments (thick lines in the half cross sections) are separated byinsulated segments (thin lines in the half cross sections).

Two implant models are described herein. They consist of a conductiveplane in which there is a

cavity. The stimulating electrode is at the bottom of the cavity,whereas the contra-electrode is a conductive plane surrounding thecavity. The two active electrode surfaces can be separated by insulatingsidewalls inside the cavity. Both electrode structures are axiallysymmetric and can thus be defined by their half cross sections (FIGS.19, (A) and (B).

The two models differ in the arrangement and the active and insulatingsurfaces inside the cavity. In one configuration, the stimulatingelectrode does not cover the entire cavity bottom; there is aninsulating section between the stimulating electrode and the cavitysidewall. In configuration B, the stimulating electrode covers theentire cavity bottom and extends onto the lower part of the cavity wall.In both configurations, the contra-electrode extends from the topsurface onto the sidewalls in the cavity.

The half cross sections can be defined using five parameters, p1-p5,which are the lengths of individual segments constituting the modelgeometries. Electrically active electrode surfaces are the thicksegments, while the insulating surfaces are the thin segments.

For configuration A, these five parameters are:

(p1) stimulating electrode radius, (p2) distance from stimulatingelectrode edge to the cavity sidewall, (p3) the length of the insulatingsegment on the cavity sidewall, (p4) the length of the contra-electrodesegment that extends onto the cavity sidewall and (p5) the length of thehorizontal segment of the contra-electrode.

For configuration B, these parameters are: (p1) the length of thehorizontal segment of the stimulating electrode, (p2) the length of thestimulating electrode segment that extends onto the cavity sidewall,(p3) distance from the stimulating electrode to the contra-electrode,(p4) the length of the contraelectrode segment extending onto the cavitysidewall and (p5) the length of the horizontal segment of thecontra-electrode.

In configuration A, the stimulating electrode is at the bottom of thewell (parameter p1) surrounded by an isolating layer (p2), while thereturning grid is at the surface of the implant (p5), which can beextended on the descending well wall (parameter p4).

In configuration B, the stimulating electrode and the returning grid canboth extend on the well wall with parameters p2 and p4, respectively.

The electrodes are micro fabricated using a classical silicon wetetching process which is why the inclination of the cavity sidewall wasfixed to the etching angle (54.7°) for (1 0 0) silicon.

Finite-element models may be created using the Comsol MultiphysicsModeling and Simulation software package (Comsol AB). The physical modelused in the simulations is the two-dimensional axially symmetric DCconductive media model, defined by the following relationships:

J=σE, ∇J=Q, Q=−∇(σ∇V),

where J is the current density vector, E is the electric field vector, σis the electric conductivity of the media, Q is the electric charge andV is the electric potential.

Electrode geometries are optimized using MATLAB (The Mathworks) to findoptimal model parameters that yield the best stimulation selectivity.Optimizations are performed under the additional constraints that thefabrication mask openings, corresponding to the electrically activeelectrode surfaces, must be at least 5 μm apart, and the depth of thecavity does not exceed 50 μm. Implant with optimized parameterscorresponding to the well depth of 30 μm and the well diameter of 30 μm(measured at the electrode array common plane level) were fabricated.

Selectivity improves with increased cavity depth. With depths up to 20μm (corresponding to the lower edge of the stimulation target zone),there is little difference between the two configurations A and B

Electrode selectivity reaches its peak at the depth of 30 μm for bothconfigurations. Configuration B yields better selectivity thanconfiguration A for all stimulating electrode sizes.

The difference is most prominent for smaller stimulating electrodesizes, e.g. for p1=5 μm, there is approximately a threefold increase inselectivity when optimal geometry B is compared to optimal geometry A,and a tenfold increase when optimal geometry B is compared to theoptimal planar electrode structure.

To eventually pack as many electrodes as possible in a limited space, itis of interest to have parameters p1 and p5 as small as possible.

This will result in having the minimal size of a single electrode andtherefore also the minimal center to-center inter-electrode distance inan array.

Example 4-1 Finite-Element Models of Four Variations of a 25×25Stimulating Electrode Array Inside a Retinal Prosthetic System

Finite-element models of four variations of a 25×25 stimulatingelectrode array inside a retinal prosthetic system were prepared: (i) aplanar array with a common counter electrode with the shape of a gridsurrounding the stimulating electrodes, (ii) a planar electrode arraywith a distant counter electrode, (iii) a three-dimensional electrodearray with a simulation electrode surrounded by a grid again serving asa counter electrode; and (iv) a 3D electrode with a distant counterelectrode. All were coated according to the present invention.

The inter-electrode distance was kept at 100 μm and the well depth forthe 3D models was 30 μm. All other model parameters were the same asdescribed in (Djilas et al, 2011) or in WO2011135273 (A1) which methodsand processes are incorporated herein by reference

For simulation purposes an image of Abraham Lincoln was then cropped anddown sampled into a square 25×25 image and its color palette reducedfrom 256 to 3 colors: white, gray, and black (see

FIGS. 13 (A) and (B)). This resulting image was then mapped into afinite-element models by assigning current densities to the stimulatingelectrodes that are proportional to the colors in the cropped image:zero for white, 1000 A/m² for gray and 2000 A/m² for black.

Example 4-2 Implant Fabrication

The implant templates used in this study were made of polyimide. Some ofthe implants were fabricated alternatively using boron doped diamond(BDD) or platinum electrodes for comparison purposes. A few otherimplants were left blank without electrodes.

The implants including BDD electrodes were fabricated as follows:silicon moulds were prepared using KOH wet etching: in order to achievetruncated pyramids, a specific design was needed, where a structure wasadded to compensate the etching speed of the 110 and 100 orientedcrystalline plans. The process was stopped when the cavities of thesilicon moulds were typically reaching 30 μm in height. A final mould isshown in FIG. 15.

FIG. 14 shows an example of Electrode modeling. This observation isconfirmed by the panels showing the side views of stimulated planarelectrodes on which there seems to be more crosstalk between twoneighboring electrodes. Indeed, this crosstalk is much reduced with the3D electrodes, and the pixels on the images generated with the 3Dconfigurations present a higher resolution (Djilas et al, 2011).

A specific process allowing the selective growth of BDD electrodes ontothe substrate was used as described elsewhere (Girard et al., 2009).This process consists of depositing seeds of nano-diamond (approximately5 nm in diameter) over the substrate. Then an aluminum mask is sputteredover the substrate and patterned by photolithography. Finally theunprotected nano-diamond is etched away by reactive ion etching (RIE)under oxygen plasma and the aluminium mask removed by wet etching. AMicrowave Plasma Enhanced Chemical Vapour Deposition (MPECVD) reactor isused to synthesize the boron-doped diamond in a gas mixture of methane(CH₄) and hydrogen (H₂) and trimethyl boron (TMB) using growthconditions as described elsewhere (Kiran et al., 2012, 12, 7669).

The thickness of the diamond layer obtained was approximately 300 nm andthe doping level was in the order of 2.5×10²¹ boron at·cm⁻³.

In a particular embodiment, the thickness of the diamond layer obtainedis comprised from less than 500 nm, preferably less than 300 nm and morepreferably is comprised from 300 to 180 nm.

The doping level is from 0.01 to 100×10²¹ boron at·cm⁻³, preferably from1 to 10×10²¹ boron at·cm⁻³ and more preferably is 2.5×10²¹ boronat·cm⁻³.

Polyimide (PI 2611) was then spin coated over the substrate so that a 10μm-thick layer of polymer was obtained. The polyimide was then cured at450° C. under nitrogen flow for 6 hours, and a 500 nm-thick aluminiumfilm was sputtered. Finally AZ4562 (Clariant, Muttenz, Switzerland)thick photoresist is spin coated on the wafer to define the implantshape, after the development the wafer was placed in Chlorine RIE toetch the aluminium layer. The unmasked polymer was etched away by O₂ RIEin order to achieve the final shape of the implant. The wafer wasimmersed in hydrofluoric acid (HF) in order to etch the sacrificialoxide layer and release the individual implants. Finally the implantswere rinsed in DI water and dried.

The process used to prepare the implant with platinum electrodes wassimilar. Here the step of BDD electrode deposition was replaced by adeposition of Pt electrodes sputtered over the substrate and patternedby standard photolithography. A third series of implants was prepared inthe same way except that no electrodes were deposited on the implants atall.

Electron microscopy imaging was performed using a Field EmissionScanning Electron Microscope (SEM) ZEISS Supra-40, operating with anacceleration voltage of 2 kV. The implants were imaged by SEM after theimplantation period. In order to perform SEM imaging, the retina/implantensembles were fixed in paraformaldehyde, the retina was peeled off, andthe implants were dehydrated in ethanol baths of increasingconcentrations in water (50%, 70%, 90% and 100% ethanol).

Example 4-2 Surgical Procedure & In Vivo Imaging

Homozygous P23H rats (Lewin et al., 1998; Machida et al., 2000)

were housed with a 12 h dark/light cycle with food and water availablead libitum. All Experiments were carried out in accordance with theEuropean Community Council Directives (86/609/EEC) and with the ARVO(Association for Research in Vision and Ophthalmology) statement for theUse of animals in ophthalmic and visual Research. Animals weresacrificed by CO₂ sedation and cervical dislocation, and all effortswere made to minimize suffering.

The surgical procedure used to implant the prototypes was described indetail previously (Salzmann et al., 2006). Briefly, P23H blind rats wereanesthetized by intraperitoneal injection with a 4:1 mixture ofketamine-xylazine (ketamine 100 mg kg⁻¹, xylazine 10 mg kg⁻¹) (Ketamine500: Virbac, Carros, France; xylazine 2%: Rompun®, Bayer Pharma,Puteaux, France). A small radial sclerotomy (length 1.5 mm) was madebehind the limbus with a slit knife. Viscoat® Intraocular viscoelasticInjection (Alcon Laboratories, Hünenberg, Switzerland) was injected witha 27 G canula into the subretinal space through the sclerotomy in orderto obtain a localised retinal detachment. After inducing a retinaldetachment, the implant was inserted into the subretinal space.Immediately after surgery, the correct position of the implant wascontrolled in vivo with indirect ophthalmoscopy (frost and lens).

In vivo imaging was performed one week after surgery and six weekslater, right before the sacrifice to observe the eye fundus byendoscopy. A MicronIII numerical endoscope (Phoenix ResearchLaboratories, Pleasanton, Calif.) was used for imaging the eye fundus,with the StreamPix V software with rat probe.

Example 4-3 Immunostaining, Confocal Imaging and Quantification

After 6 to 8 weeks, animals were sacrificed by CO₂ sedation and cervicalelongation. The eyes were removed, put in phosphate buffer saline (PBS,0.1 M, pH 7.4) and dissected to keep only the tissue fragment containingthe implant. This fragment was fixed overnight at 4° C. in PBScontaining paraformaldehyde (4% wt/vol) and then rinsed in PBS.

For immunolabelling, retina fragments were incubated in a blockingsolution (PBS containing 10% bovine serum albumin (Sigma, St. Louis,Mo.), 1% Triton X-100 (Sigma), 0.5% Tween 20 (Sigma) and 0.1 g/LThimerosal (Sigma)) for 1 h at room temperature (room temperature isfrom 22° C. to 25° C., in particular 20° C.). Primary antibodies wereincubated for 2 days at room temperature in the blocking solution(dilution 1:2). Polyclonal antibodies were directed against chicken GFAP(1:100, LSBio, Seattle, Wash.) and rabbit PKCα (C-20) (1:1000, SantaCruz Biotechnology, Dallas, Tex.). Monoclonal antibody was directedagainst mouse Goα (1:200, Chemicon, Darmstadt, Germany). The fragmentswere rinsed and then incubated with secondary antibodies, goatanti-chicken IgG, goat anti-mouse IgG and goat anti-rabbit conjugated toAlexa™ 633, Alexa™ 594 and Alexa™ 488 respectively (1:500, MolecularProbes, Invitrogen, Eugene, Oreg.) for one day. Cell nuclei wererevealed with 4′,6-Diamidino-2-phenylindole (DAPI) which was addedduring the final incubation period.

The implant/retina ensemble was then rinsed and mounted with PermanentMounting medium (MMFrance) on a microscope slide to be visualized underan upright confocal microscope (Olympus, France).

The presence of bipolar cells within the wells of 3D-structured implantswas quantified as the ratio of bipolar cells over the total number ofcell nuclei per well for each material and was countedsemi-automatically with Imaris software (Bitplane AG, Zurich,Switzerland).

Example 4-4 Statistical Analysis

The results from 3 polyimide implants, 5 diamond implants and 3 platinumimplants, taking the values from 4 to 9 wells per implant forquantification were collected. All data are expressed as means±s.e.m.Gaussian distribution of raw data was tested with Shapiro-Wilk normalitytest. A one-way ANOVA was used for variance analysis, followed in caseof significance by either a Bonferroni post-hoc test (Gaussiandistribution) or a Dunns post-hoc test (no Gaussian distribution) tocompare the means of each group. Differences were considered significantat *p<0.05, **p<0.01 and ***p<0.001.

Example 4-5 Diamond Electrodes on a Flexible Foil

The possibility to fabricate flexible substrates that adapt to thecurvature of soft tissues such as the retina is essential. Synthesistechniques enabling the growth of diamond rely on high temperatures andmicrowave plasma techniques that are not compatible with most types ofsoft substrates.

It is one object of the present invention to provide a substrate thatadapts to the curvature of soft tissues.

The solution we developed is based on a lift-off process for which asoft polymer deposited on top of a patterned diamond layer is used toprovide support to the diamond layer. A soft polymer deposited on top ofa patterned diamond layer supporting a diamond layer, is provided.

It is within the scope of the person skilled in the art to determine thesoftness or hardness of polymer and of devices according to theinvention. For example, a high resolution nanoindenter or Depth-SensingIndentation (DSI) technique may be used, to evaluate mechanicalproperties of materials at the nanoscale level, especially hardness.

This process is displayed on FIG. 15 (A) and the device is fabricatedbased on the mask shown in FIG. 15 (B).

In brief, this was made feasible from the selective growth of thediamond electrodes on a rigid sacrificial substrate (silicon mould onFIG. 15 C), on which is prepared the polyimide structure, which isreleased from the substrate at the end of the process. The approachenables large dimensions to be fabricated as visible on FIG. 15 D.

A process comprising a selective growth of a diamond electrode on asubstrate, in particular a silicon mould, preparing a polyimidestructure, optionally releasing said polyimide structure from thesubstrate.

Example 4-6 In Vivo Imaging

Soft polyimide implants with diamond and metal electrodes were insertedinto the subretinal space of P23H rats (animal model of photoreceptordegeneration) for 6 to 8 weeks, to assess the tissue reaction. Thecorrect implant insertion was controlled in vivo using a MicronIIInumerical endoscope, see fundus images on 16. The presence of retinalblood vessels above the device confirmed the subretinal position of theimplants. This examination also enabled us to visualize the resorbtionof the subretinal bleb generated to introduce the subretinal implant.

Example 4-7 Characterization of Diamond Implants

The original fabrication process enabled fabricating soft implantsexhibiting several 3D wells either uncoated or coated with eitherdiamond or Platinum. In the case of diamond the coating covers the wholearea on the implant visible on the photograph FIGS. 17 B and E,including the walls and the bottom of the cavities, whereas in the caseof platinum the metal coverage appears in light grey color in panels Cand F. After 6 to 8 weeks of implantation, the surface of the implantedelectrodes was observed by Scanning Electron Microscopy (SEM) in orderto assess the physical stability of the implants. The results are shownin FIG. 17 for the three materials tested. On the polyimide implant (A,D), the surface of the material is similar to before implantation, withno visible defect. Note that the white trace visible on FIG. 17 A is dueto charge accumulation on this insulating surface during SEM imaging.Similarly, the diamond films (B, E) showed no discontinuities and propersurface coverage, thus suggesting a good adhesion of diamond ontopolyimide. Those diamond surfaces appeared very smooth as opposed toconventional polycrystalline BDD. This is because the way the implantsare fabricated, the side of diamond that is actually exposed to thetissues is the smooth face that was originally in contact with thesilicon surface.

Finally on the metallic implants (C, F), the light grey areascorresponding to platinum appear also free of significant defects anddarker because of organic matter (remaining cells). Hence the surface ofboth diamond and platinum has not been damaged during the implantationperiod. For all these implants, some cells or tissue remain visible onthe implants and are particularly concentrated inside the cavities onthe enlarged views (D-F). To validate the presence of retinal neurons,particularly bipolar cells, we have investigated the nature of theseremaining cells within the 3D structures by immunostaining and confocalimaging. The results are shown in the following section.

Example 4-8 Bipolar Cell Migration

To study the interface between a tissue and a prosthetic device,sections are commonly prepared with or without removing the device priorto sectioning. However, although this histological procedure could beperformed in previous studies on polyimide prototypes (Salzman et al.,2006; Djilas et al., 2011), the presence of metallic as well as BDDelectrodes makes this approach unrealistic without degrading theobserved samples. In fact, according to the mechanical stiffness of someof the polyimide prototypes, the cryosections of the implants frequentlyinduced the dissociation of the samples as previously illustrated(Djilas et al., 2011). Greater difficulties were anticipated with thepresence of metal or diamond electrodes on the implants. Therefore, topreserve the biological samples, an innovative approach based onconfocal microscopy was developed and enabling the direct observation ofthe tissue/implant interface. Retinal bipolar neurons and glial cellswere immunolabelled as a wholemount preparation containing both theretina and the implant. The immunostaining protocol was adapted topreserve this interaction while allowing antibody diffusion within theretinal tissue over a 100 μm distance. FIG. 18 illustrates such confocalimages of the tissue/implant interface for a diamond implant (A-B),along with the image processing for cell quantification (C-E).

Cell nuclei were labeled by the DAPI stain (blue) while glial cells andretinal bipolar neurons were immunolabelled by the GFAP antibody (grey)and the Goα and PKCα antibodies (green and red), respectively. The ×20magnification view of the retina/implant wholemount (A) allowsvisualizing the presence of nuclei stain in all 25 cavities. The ×40magnification images (panel B) allows a better observation of themorphology of bipolar cells filling the cavities. The vertical views(x-z axes) similar to vertical retinal sections showed retinal bipolarneurons in green filling the entire cavity up to the bottom. Thehorizontal views (x-y axes) confirmed that the bipolar neurons werepresent at high densities within each well regardless of the materials.

To further assess the material biocompatibility, the number of cellnuclei was quantified in the cavities (D) as well as the percentage ofbipolar neurons versus all cell nuclei in each cavity (E). Thisquantification required to isolate all pixels in a single well, todefine in this volume the spheres corresponding to the immunolabellingof bipolar cell nuclei and to count them (C). FIG. 18 illustrates thesecell counts of all ON bipolar cells in the wells with differentmaterials. These images provide evidence that the residual retinaremains very plastic to mould itself into the 3D implant wells.Furthermore, the absence of a massive fibrotic inflammatory reaction butrather the presence of bipolar neurons in the wells suggests that thedifferent materials according to the invention are not toxic to neurons.

Conclusion:

The results with our 3D prototypes inserted in the subretinal space ofP23H rats showed that the retina shapes into the wells with bipolar cellneurons fully integrating the inside of the cavities. The presentinvention provides a physical confinement of the stimulus. Theseexperiments also indicated an increased stability of the tissue/implantinterface with 3D prototypes than with planar implants. As aconsequence, stimulating bipolar cells in such a well would activate aretinal column independent from the neighboring retinal columnsactivated by the neighboring wells. Our three-dimensional design of thesubretinal implants would therefore allow considering each electrode asan independent pixel.

Our results show the compatibility of diamond with flexible implantfabrication, the low tissue scarring in contact with diamond electrodesand the possibility to selectively stimulate neurons in the vicinity ofeach electrode independently from the neighboring electrodes. Our studyclearly demonstrates that nanocristalline diamond is safe for theretinal tissue and more generally for glial cells and neurones duringchronic implantation.

The object of the present invention is therefore a major improvement ascompared to those disclosed in prior art.

This study confirms the benefit of 3D-structured electrodes for theconception of retinal implants to tremendously improve the resolution ofthe stimulation. It also underlines the high interest diamond offers asan attractive electrode material for neuroprostheses, in particularflexible neuroprostheses.

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1-15. (canceled)
 16. A method for promoting the growth or at least thedirect interfacing of adult neurons on a material without substantiallypromoting the growth and direct interfacing of glial cells on saidmaterial, comprising contacting a material having a first biocompatiblechemically oxygen terminated or H-terminated carbon-based material,bound to a biocompatible second chemically oxygen terminated orH-terminated carbon-based material, said first and said secondcarbon-based material forming an electrode, said first material being inparticular selected from the group consisting of nanocrystalline diamondwhich is doped by either boron, phosphorus or nitrogen, to become asemiconductor, or graphene, nanotubes, or nanotubes on diamond, thesurface of which is substantially free of any peptide coating, saidfirst carbon-based material having an electrical conductivity of atleast about 0.01 S·cm⁻¹, said second biocompatible chemically oxygenterminated or H-terminated carbon-based material, being identical ordifferent from said first carbon-based material, said secondcarbon-based material presenting a peptide coating, in particular apeptide from the extracellular matrix, to promote the growth and atleast the direct interfacing of adult glial cells, said secondchemically oxygen terminated or H-terminated carbon-based material beingin particular selected from the group consisting of nanocrystallinediamond which is doped in particular with boron, in particular aboron-doped hydrogen terminated diamond or oxidized diamond, graphene ornanotubes, in particular diamond grafted nanotubes, with a tissuecomprising adult neurons.
 17. The method according to claim 16, whereinsaid adult neurons are found in tissues selected from the groupconsisting of the retina, thalamus, cortex, vestibular system, cochlea,brain stem, midbrain, colliculus, subthalamic nucleus, globus pallidusinterna, zona incerta, pallidofugal fibers, periaqueductal gray,periventricular gray, internal capsule, ventral posterolateral nucleusand ventral posteromedial nucleus, subgenual cingulate gyrus, nucleusaccumbens, ventral capsule/ventral striatum, inferior thalamic peduncle,lateral habenula vagus nerve, afferent nerves, spinal cord, large dorsalcolumns, nerves controlling muscle activity such as those for locomotionor sphincter opening.
 18. The method according to claim 16, wherein saidelectrode is shaped like a penetrating electrode, such as a penetratingneedle, in particular a tri-dimensional needle, or is positioned on apenetrating support, which can be shaped like a needle, in particular atri-dimensional needle, and in particular, a plurality of electrodesbeing combined to form an electrode array, and more particularly saidelectrode or electrode array is fixed on a support to form an implant ora prosthesis, in particular wherein the length of said penetratingneedle and/or electrode is comprised from about 10 μm to about 1 to 2 cmand the diameter is comprised from 20 μm to about 500 μm.
 19. The methodaccording to claim 16, wherein said first chemically oxygen terminatedor H-terminated carbon-based material constitutes the tip of anindividual penetrating electrode or the penetrating electrode tips ofelectrode arrays and said second carbon-based material constitutes theinterfacing surface between the prosthesis or implant and the glialsurface of any neuronal structure.
 20. The method according to claim 16,wherein said peptide is from the extracellular matrix, in particularselected from the group consisting of poly-lysine, in particularpoly-D-lysine, poly-ornithine, laminin or combination thereof, inparticular the thickness of the peptide coating being comprised fromabout 0.5 μg/cm² to about 5 μg/cm².
 21. An electrode comprising a firstchemically oxygen terminated or H-terminated carbon-based material topromote the growth or at least the direct interfacing of adult neurons,without substantially promoting the growth and direct interfacing ofglial cells on said material, which is bound to a second biocompatiblechemically oxygen terminated or H-terminated carbon-based material,identical or different from said first carbon-based material, such asdefined in claim 16, said second carbon-based material presenting apeptide coating, in particular a peptide from the extracellular matrix,to promote the growth and at least the direct interfacing of adult glialcells, in particular said first and second carbon-based material areselected from the group consisting of nanocrystalline diamond which isdoped by either boron, phosphorus or nitrogen, to become asemiconductor, or graphene, nanotubes, or nanotubes on diamond, and moreparticularly, said first chemically oxygen terminated or H-terminatedcarbon-based material and said second chemically oxygen terminated orH-terminated carbon-based material are similar or identical. 22.Electrode according to claim 21, wherein said electrode is shaped like apenetrating electrode, such as a penetrating needle, in particular atri-dimensional needle, or is positioned on a penetrating support, whichcan be shaped like a needle, in particular a tri-dimensional needle, inparticular a plurality of electrodes and is combined to form anelectrode array, and more particularly said electrode or electrode arrayis fixed on a support to form an implant or a prosthesis.
 23. Electrodeaccording to claim 21, wherein said first carbon-based materialconstitutes the tip of an individual penetrating electrode or thepenetrating electrode tips of electrode arrays and said secondcarbon-based material constitutes the interfacing surface between theprosthesis or implant and the glial surface of any neuronal structure.24. Electrode according to claim 22, wherein said peptide is selectedfrom the group consisting of poly-lysine, in particular poly-D-lysine,poly-ornithine or laminin, in particular, the thickness of the peptidecoating is comprised from about 0.5 μg/cm² to about 5 μg/cm², and moreparticularly the length of said penetrating needle and/or electrode iscomprised from about 10 μm to about 1 to 2 cm and the diameter of saidelectrode is comprised from 20 μm to about 500 μm.
 25. Process ofpreparation of an electrode or needle according to claim 21, comprisingthe following steps: a. preparation of conductive biocompatiblechemically oxygen terminated or H-terminated carbon-based materialscaffold, b. optionally, oxidation of said H-terminated carbon-basedmaterial scaffold to obtain a first carbon-based material, c. optionallydoping a second part of said chemically oxygen terminated orH-terminated carbon-based material scaffold, in particular with boron,phosphorus or nitrogen, to obtain a partially doped second part of saidchemically oxygen terminated or H-terminated carbon-based materialscaffold, d. coating said second part of said chemically oxygenterminated or H-terminated carbon-based material scaffold with apeptide, optionally doped, in particular a peptide from theextracellular matrix, to obtain a second chemically oxygen terminated orH-terminated carbon-based material, said first chemically oxygenterminated or H-terminated carbon-based material and said secondchemically oxygen terminated or H-terminated carbon-based materialconstituting an electrode, in particular said peptide is selected fromthe group consisting of poly-lysine, in particular poly-D-lysine,poly-ornithine or laminin.
 26. Process according to claim 25, whereinsaid first part of said chemically oxygen terminated or H-terminatedcarbon-based material scaffold is doped with boron, phosphorus ornitrogen, in particular said first part of H-terminated carbon-basedmaterial scaffold is oxidized.
 27. Process according to claim 25,wherein said first part of chemically oxygen terminated or H-terminatedcarbon-based material is nanocrystalline diamond, polycrystallinediamond, or graphene.
 28. Process according to claim 25, wherein saidsecond part of chemically oxygen terminated or H-terminated carbon-basedmaterial is nanocrystalline diamond, polycrystalline diamond, orgraphene, in particular said first and/or second chemically oxygenterminated or H-terminated carbon-based material is different frommonodispersed nanodiamond powder on nanocrystalline diamond or onpolycrystalline diamond or from nanocrystalline diamond within whichnanotubes are partially embedded.
 29. A method of implementing animplant or a prosthesis liable to promote the growth of adult neurons orat least the direct interfacing of adult neurons and to stimulate saidadult neurons, in particular said neurons are retinal neurons and saidimplant is a retinal implant, and/or in particular said electrodefurther comprises glial cells comprising using an electrode as definedin claim
 16. 30. The method according to claim 29, wherein saidelectrode is (i) a planar array with a common counter electrode with theshape of a grid surrounding the stimulating electrodes, (ii) a planarelectrode array with a distant counter electrode, (iii) athree-dimensional electrode, in particular a three-dimensional electrodearray with a simulation electrode surrounded by a grid again serving asa counter electrode; or (iv) a three-dimensional electrode with adistant counter electrode with a inter-electrode distance was kept at100 μm and the well depth for the 3D models was 30 μm.